Synthetic metabolic funneling for biochemical production

ABSTRACT

Certain embodiments provide a method for preparing a biochemical product (e.g., phenol, catechol, or muconic acid, or a salt thereof). For example, such methods include contacting a recombinant host having two or more recombinant pathways with a fermentable carbon source and growing the recombinant cell for a time sufficient to synthesize the product. In certain embodiments, each recombinant pathway: 1) is capable of producing the same final biochemical product; 2) comprises at least one gene encoding a polypeptide; 3) is derived from a different endogenous metabolite as its immediate precursor; and 4) converges to the same final product or the same intermediate metabolite.

RELATED APPLICATION

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 62/533,997 filed on Jul. 18, 2017, whichapplication is incorporated by reference herein.

BACKGROUND

In recent years, cis,cis-muconic acid (hereafter also referred to asmuconic acid, muconate, cis,cis-muconate, or MA) has continued to emergeas a diacid bioproduct of industrial interest, most notably due to itspotential role as a precursor to adipic acid—a platform chemical usedfor the synthesis of various plastics and polymers (e.g., Nylon 6,6)(Adkins, et al., 2012. Frontiers in microbiology. 3, 313; Deng, et al.,2016. Biochem Eng J. 105, 16-26). Through metabolic pathway engineering,numerous studies have reported the engineering of microbes capable ofproducing MA as the focal product from renewable feedstocks (Curran etal., 2013. Metabolic engineering. 15; Draths, K. M., Frost, J. W., 1994.et al., Journal of the American Chemical Society. 116, 399-400; Lin, etal., 2014. Metabolic engineering. 23; Niu, et al., 2002. Biotechnologyprogress. 18, 201-11; Sengupta, et al., 2015. Applied and environmentalmicrobiology. 81, 8037-8043; Sun, et al., 2013a. Applied andenvironmental microbiology. 79; Sun, et al., 2013b. Applied andenvironmental microbiology. 79, 4024-30; Weber, et al., 2012b. Appliedand environmental microbiology. 78; Zhang, et al., 2015a. Microb CellFact. 14, 134; Zhang, et al., 2015b. Proceedings of the National Academyof Sciences of the United States of America. 112, 8266-71). Draths andFrost were first to report MA biosynthesis from glucose in Escherichiacoli, following construction of a three-step pathway stemming fromendogenous 3-dehydroshikimate (3DHS), a key intermediate in the shikimicacid pathway (Draths, K. M., Frost, J. W., 1994. et al., Journal of theAmerican Chemical Society. 116, 399-400). Said pathway, which has sincealso been functionally re-constructed in Saccharomyces cerevisiae(Curran et al., 2013. Metabolic engineering. 15; Weber, et al., 2012a.Applied and environmental microbiology. 78, 8421-30), proceeds throughthe intermediates protocatechuate (PCA) and catechol via 3-DHSdehydratase, PCA decarboxylase, and catechol 1,2-dioxygenase (hereafterreferred to as ‘3DHS-derived’ pathway or pathway MA1). Achievingsignificant MA production via this pathway has typically requireddeletion of aroE (encoding shikimate dehydrogenase, an essential gene inminimal media). This mutation results in auxotrophies for the aromaticamino acids phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp),as well as the growth essential vitamins p-aminobenzoate (pAB),p-hydroxybenzoate (pHBA), and 2,3-dihydroxybenzoate (2,3-DHB)—all ofwhich are derived from chorismate as their last common precursor. Thus,although high MA titers and yields have been achieved by expressing thispathway in an E. coli ΔaroE background (2.4 g/L at 0.24 g/g-glucose,respectively, in shake flasks (Draths, K. M., Frost, J. W., 1994. etal., Journal of the American Chemical Society. 116, 399-400), and 36.8g/L at 0.17 g/g-glucose in a fed-batch bioreactor (Niu, et al., 2002.Biotechnology progress. 18, 201-11)), doing so required each of theabove six nutrients or shikimate to first be supplemented into theminimal salts media—an expensive and poorly scalable practice.

Accordingly, new compositions and methods for generating MA are needed.New compositions and methods are also generally needed for generatingother biochemical products, including phenol and catechol.

SUMMARY

Described herein is a modular approach and methods for the microbialproduction of biochemical/biofuel products, such as phenol, catechol,and muconic acid, from renewable substrates using recombinantmicroorganisms. Phenol and catechol are aromatic building blockchemicals, while muconic acid is used as precursor in plasticsproduction. Biosynthesis of each of the three products was achieved byengineering and expressing a series of non-natural, modular enzymepathways. In contrast to existing methods for the bioproduction ofphenol, the methods described herein are not subject to equilibriumlimitations or feedback inhibition of pathway enzymes. Meanwhile, incontrast to existing methods for the bioproduction of catechol andmuconic acid, the methods described herein benefit from an improvedthermodynamic driving force and strain engineering strategies withimproved host compatibility and sustainability.

Accordingly, certain embodiments provide a method for preparing abiochemical product (e.g., phenol, catechol, or muconic acid, or a saltthereof). For example, such methods include contacting a recombinanthost having two or more recombinant pathways with a fermentable carbonsource and growing the recombinant cell for a time sufficient tosynthesize the product. In certain embodiments, each recombinantpathway: 1) is capable of producing the same final biochemical product;2) comprises (or consists of) at least one gene encoding a polypeptide;3) is derived from a different endogenous metabolite as its immediateprecursor; and 4) converges to the same final product or the sameintermediate metabolite.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Five non-natural pathways engineered for muconic acidbiosynthesis from glucose. Black arrows represent native E. colienzymatic steps; gray arrows represent heterologous steps; dotted arrowsrepresent multiple enzymatic steps. Δ_(r)G′° is the change in Gibbs freeenergy due to reaction as determined using the online tool eQuilibrator(http://equilibrator.weizmann.ac.il) at a reference state of 25° C., pH7, and ionic strength of 0.1 M. A: 3-dehydroshikimate dehydratase, B:protocatechuate decarboxylase, C: catechol 1,2-dioxygenase, D:chorismate pyruvate lyase, E: p-hydroxybenzoate hydroxylase, F:p-hydroxybenzoate decarboxylase, G: isochorismate synthase, H:isochorismate pyruvate lyase, I: salicylate decarboxylase, J: tyrosinephenol lyase, K: phenol hydroxylase.

FIG. 2. Comparing the predicted change in Gibbs free energy of reaction)(Δ_(r)G′°) as a function of pathway progress from 3-DHS (the last commonendogenous precursor) to MA for each of the five pathways studied (MA1:squares, MA2: circles, MA3: triangles, MA4: stars, MA5: diamonds). Alsocompared are the predicted maximum MA yields (Y_(P/S)), MA yields withgrowth (Y_(P/S+growth)), and biomass yields (Y_(X/S)) from glucose, asdetermined by elementary flux mode (EFM) analysis. Note: Yields are thefraction of carbons in the substrate (glucose) that end up in theproduct (MA). PCA: protocatechuate, Tyr: tyrosine, pHBA:p-hydroxybenzoate.

FIGS. 3A-3B. Screening candidate pathway enzymes using whole restingcells supplemented with (dashed lines, open symbols) and without (solidlines, filled symbols) 100 μM Fe(NH₄)₂(SO₄)₂ supplementation. FIG. 3A)Conversion of phenol (squares) to catechol (circles) by E. coli BW25113pPh cultures. FIG. 3B) Conversion of phenol (squares) to catechol(circles) or muconic acid (diamonds) by E. coli BW25113 pPh-CatAcultures. Error bars represent one standard deviation from triplicateexperiments.

FIG. 4. Detection of 1,2,3-trihydroxybenzene (1,2,3-THB) in culturesupernatants from E. coli BW25113 whole resting cells expressingphKLMNOP of Pseudomonas stutzeri OX1. Cells were suspended in PBS buffer(pH 6.8) supplemented with 1 mM phenol.

FIGS. 5A-B. FIG. 5A. Engineering a ‘synthetic funnel’ for MA productionvia the simultaneous co-expression of two distinct biosynthesispathways, stemming from two different precursors (3DHS and chorismate),both derived from the shikimic acid pathway. Also shown are the nativemechanisms by which different aromatic degradation pathways also‘funnel’ various substrates and intermediates towards MA, in this casefor further catabolism. Black bold dashed arrows represent multipleenzyme reactions native to E. coli; solid black arrows representindividual enzymes steps associated with the pathways engineered in thisstudy; Gray solid and dashed arrows represent individual and multi-stepenzyme processes associated with the aerobic degradation of aromaticchemicals. PCA, protocatechuate; pHBA, p-hydroxybenzoate; AAA, aromaticamino acids; TCA, tricarboxylic acid cycle. FIG. 5B. Engineering a‘synthetic funnel’ for MA production via the simultaneous co-expressionof two distinct biosynthesis pathways, namely MA1 (composed of steps A,B, C) and MA5 (composed of steps D, E, B, C). The combined, ‘funneling’pathway MAF (composed of steps A, B, C, D, E) produces MA bysimultaneously drawing from two different precursors (3DHS andchorismate), both derived from the shikimic acid pathway. Black arrowsrepresent native E. coli enzymatic steps; gray arrows representheterologous steps; dotted arrows represent multiple enzymatic steps.Δ_(r)G′° is the change in Gibbs free energy due to reaction asdetermined using the online tool eQuilibrator(http://equilibrator.weizmann.ac.il) at a reference state of 25° C., pH7, and ionic strength of 0.1 M. “A”: 3-dehydroshikimate dehydratase,“B”: protocatechuate decarboxylase, “C”: catechol 1,2-dioxygenase, “D”:chorismate pyruvate lyase, “E”: p-hydroxybenzoate hydroxylase.

FIG. 6. Three non-natural pathways engineered for phenol production fromglucose. Black arrows represent enzyme steps native to E. coli whereasgray arrows are heterologous, dotted arrows represent multiple enzymaticsteps. Δ_(r)G′° is the change in Gibbs free energy due to reaction asdetermined using the online tool eQuilibrator(http://equilibrator.weizmann.ac.il) at a reference state of 25° C., pH7, and ionic strength of 0 M. A: tyrosine phenol lyase, B: isochorismatesynthase, C: isochorismate pyruvate lyase, D: salicylate decarboxylase,E: chorismate pyruvate lyase, F: p-hydroxybenzoate decarboxylase.

FIG. 7. Engineering a synthetic ‘metabolic funnel’ for enhanced phenolproduction. The synthetic ‘metabolic funnel’ was constructed via thesimultaneous co-expression of two or three distinct pathways, allstemming from chorismate as the last common endogenous precursor. Blackbold dashed arrow represents multiple enzyme reactions native to E.coli; solid arrows represent individual enzyme steps associated with theengineered pathways; pHBA: p-hydroxybenzoate.

DETAILED DESCRIPTION

By rational re-engineering of their metabolism, microorganisms can beengineered to convert biomass-derived feedstocks to specific focalproducts of interest, including chemicals and fuels traditionallyderived from petroleum. In such cases, metabolic engineering can beapplied not only to improve the function of naturally-occurringpathways, but also for the de novo creation of synthetic pathways toenable the novel biosynthesis of non-inherent and even non-naturalproducts. Traditional applications of metabolic pathway engineeringfocus on expressing in a recombinant host a single pathway that has beendesigned and engineered for the biosynthesis of a specific and singlefocal product of interest. In certain cases, however, there are morethan one possible enzyme pathways that have been engineered, proposed,or hypothesized to enable biosynthesis of the same product molecule.Common examples of this include, but are not limited to, ethanol,3-hydroxypropionate, phenol, catechol, muconic acid, and isoprenoids.Typically, different pathways offer their own unique and advantages aswell as disadvantages relative to the other distinct pathway options.Examples include, but are not limited to: differences in product titerand/or yield; differences in host toxicity owing to the involvement ofinhibitory genes, proteins, or intermediate metabolites; differences inhost fitness owing to competition for different growth essentialprecursor metabolites; and, differences in thermodynamic driving forces.In light of the foregoing, it would be an advancement to provide amethod by which to balance the relative trade-offs between differentalternative metabolic pathways engineered for the biosynthesis of thesame product.

Accordingly, as described herein, certain embodiments comprise an invivo method for the production of a biochemical via a recombinant hostcell by simultaneously co-expressing two or more enzyme pathways eachengineered for the biosynthesis of the same final product (see, Thompsonet al., ACS Synth. Biol. 2018, 7(2):565-575 and supporting information,which are incorporated by reference herein in their entirety). In thisway, precursor metabolites are more effectively and extensively funneledtowards the final product of interest. Moreover, the relative advantagesand disadvantages of different pathway alternatives can be effectivelybalanced to optimize inherent trade-offs including, for example, hostfitness versus pathway flux and yield.

Certain embodiments also comprise specific methods for the in vivoproduction of phenol, catechol, and muconic acid via the aforementionedapproach. Specifically, a series of novel, modular enzyme pathways andmicroorganisms have been engineered to produce each of phenol, catechol,and muconic acid as focal products from renewable resources (e.g., froma fermentable substrate such as glucose). All three products representuseful molecular building blocks for the production of numerous fine andcommodity chemicals, as well as plastic materials. Currently, all threeproducts are derived from non-renewable petroleum feedstocks. Theproposed methods and microorganisms represent an advance over previousmethods and compositions, specifically by addressing key thermodynamic,enzymatic, and/or resource limitations associated with the conventionalbio-production of said compounds. For these three compounds,co-expression of multiple pathways in the same recombinant microorganismhas been shown to promote higher product titers and yields thanachievable by expressing any single pathway alone. Additionally, themethods described herein are further suitable for improving theproduction of other bioproducts of interest from renewable andsustainable resources.

Exemplary Methods According to One or More Embodiments

One embodiment provides a method for preparing a biochemical product,the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising two or more recombinant        pathways, wherein:        -   a) each pathway is capable of producing the same final            biochemical product;        -   b) each pathway consists of or comprises at least one gene            encoding a polypeptide (e.g., a recombinant gene);        -   c) each pathway is derived from a different endogenous            metabolite as its immediate precursor; and        -   d) each pathway converges to the same final product;    -   and ii) growing said recombinant cell for a time sufficient to        synthesize the final biochemical product.

One embodiment provides a method for preparing a biochemical product,the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising two or more recombinant        pathways, wherein:        -   a) each pathway is capable of producing the same final            biochemical product;        -   b) each pathway consists of or comprises at least one gene            encoding a polypeptide;        -   c) each pathway is derived from a different endogenous            metabolite as its immediate precursor;        -   d) each pathway converges to the same intermediate            metabolite; and        -   e) each pathway continues to the same final product;    -   and ii) growing said recombinant cell for a time sufficient to        synthesize the product.

One embodiment provides a method for preparing a biochemical product,the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, wherein the recombinant host comprises two or more        recombinant pathways, and wherein:        -   a) each pathway is capable of producing the same final            biochemical product;        -   b) each pathway consists of or comprises at least one gene            encoding a polypeptide;        -   c) each pathway is derived from the same endogenous            metabolite as its immediate precursor;        -   d) each pathway proceeds via different intermediate            metabolites;        -   e) each pathway consists of or comprises at least one gene            encoding polypeptides with differing activities; and        -   f) each pathway converges to the same final product; and    -   ii) growing said recombinant cell for a time sufficient to        synthesize the product.

One embodiment provides a method for preparing a biochemical product,the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, wherein the recombinant host comprises two or more        recombinant pathways, and wherein:        -   a) each pathway is capable of producing the same final            biochemical product;        -   b) each pathway consists of or comprises at least one gene            encoding a polypeptide;        -   c) each pathway is derived from the same endogenous            metabolite as its immediate precursor;        -   d) each pathway proceeds via different intermediate            metabolites;        -   e) each pathway consists of or comprises at least one gene            encoding polypeptides with differing activities;        -   f) each pathway converges to the same intermediate            metabolite; and        -   g) each pathway continues to the same final product; and    -   ii) growing said recombinant cell for a time sufficient to        synthesize the product.

One embodiment provides a method for the production of phenol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity; and        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce phenol.

One embodiment provides a method for the production of phenol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   b) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   c) at least one gene encoding a polypeptide having            salicylate decarboxylase activity; and        -   d) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce phenol.

One embodiment provides a method for the production of phenol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity; and        -   c) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce phenol.

One embodiment provides a method for the production of phenol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   b) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   c) at least one gene encoding a polypeptide having            salicylate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having            chorismate lyase activity; and        -   e) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce phenol.

One embodiment provides a method for the production of phenol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   b) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   c) at least one gene encoding a polypeptide having            salicylate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   e) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity; and        -   f) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce phenol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity; and        -   c) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity; and        -   b) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity; and        -   c) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   b) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   c) at least one gene encoding a polypeptide having            salicylate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity; and        -   e) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity;        -   c) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity; and        -   d) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   b) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   c) at least one gene encoding a polypeptide having            salicylate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   e) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity; and        -   f) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   c) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity; and        -   e) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   c) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   e) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   f) at least one gene encoding a polypeptide having            salicylate decarboxylase activity; and        -   g) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   c) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity; and        -   e) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   c) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity; and        -   d) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   c) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   d) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity; and        -   e) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   c) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   d) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   e) at least one gene encoding a polypeptide having            salicylate decarboxylase activity; and        -   f) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   c) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity; and        -   d) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   c) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   d) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   e) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity; and        -   f) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   c) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   d) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   e) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   f) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   g) at least one gene encoding a polypeptide having            salicylate decarboxylase activity; and        -   h) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of catechol, themethod comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   c) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   d) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   e) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity; and        -   f) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce catechol.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity;        -   c) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   d) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity;        -   b) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   c) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   c) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity; and        -   d) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   b) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   c) at least one gene encoding a polypeptide having            salicylate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity;        -   e) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   f) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity;        -   c) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity;        -   d) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   e) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   b) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   c) at least one gene encoding a polypeptide having            salicylate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   e) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity;        -   f) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   g) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   c) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity;        -   e) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   f) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   c) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   e) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   f) at least one gene encoding a polypeptide having            salicylate decarboxylase activity;        -   g) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   h) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   b) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   c) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   d) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity;        -   e) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   f) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   c) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   d) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity; and        -   e) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   c) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   d) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity;        -   e) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   f) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   c) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   d) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   e) at least one gene encoding a polypeptide having            salicylate decarboxylase activity;        -   f) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   g) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   c) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity;        -   d) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   e) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   c) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   d) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   e) at least one gene encoding a polypeptide having tyrosine            phenol lyase activity;        -   f) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   g) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   c) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   d) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   e) at least one gene encoding a polypeptide having            isochorismate synthase activity;        -   f) at least one gene encoding a polypeptide having            isochorismate pyruvate lyase activity;        -   g) at least one gene encoding a polypeptide having            salicylate decarboxylase activity;        -   h) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   i) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of muconic acid, ora salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene encoding a polypeptide having            3-dehydroshikimate dehydratase activity;        -   b) at least one gene encoding a polypeptide having            chorismate lyase activity;        -   c) at least one gene encoding a polypeptide having            p-hydroxybenzoate hydroxylase activity;        -   d) at least one gene encoding a polypeptide having            protocatechuate decarboxylase activity;        -   e) at least one gene encoding a polypeptide having            p-hydroxybenzoate decarboxylase activity;        -   f) at least one gene encoding a polypeptide having phenol            2-monooxygenase activity; and        -   g) at least one gene encoding a polypeptide having            1,2-catechol dioxygenase activity;    -   and ii) growing said recombinant cell for a time sufficient to        produce muconic acid, or a salt thereof.

One embodiment provides a method for the production of phenol, catecholor muconic acid, or a salt thereof, the method comprising:

-   -   i) contacting a recombinant host cell with a fermentable carbon        source, said recombinant host comprising:        -   a) at least one gene described herein or a combination of            genes as described herein (e.g., as described in the Figures            or Tables (e.g., Table 1));    -   and ii) growing said recombinant cell for a time sufficient to        produce phenol, catechol or muconic acid, or a salt thereof.

In certain embodiments, the recombinant host further comprises at leastone gene encoding a polypeptide having chorismate mutase/prephenatedehydrogenase activity.

In certain embodiments, the methods further comprise amplifying genomicDNA for the gene of interest (e.g., via PCR), cloning the amplifiedgenetic material into an expression vector, and transforming the hostcell to express the encoded protein.

In one embodiment, the biochemical product is phenol, catechol ormuconic acid, or a salt thereof.

Substrate/Carbon Source

The term “fermentable carbon substrate” or “fermentable carbon source”refers to a carbon source capable of being metabolized by therecombinant host cells described herein, in either its purified orunpurified form. For example, carbon sources may be selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,fats, lipids, aromatic monomers and/or oligomers, organic acids,glycerol, and one-carbon substrates, or mixtures thereof. In oneembodiment, the fermentable carbon source is selected from the groupconsisting of monosaccharides, oligosaccharides, polysaccharides,glycerol, carbon dioxide, methanol, methane, formaldehyde, formate,amino acids and carbon-containing amines.

In one embodiment, the fermentable carbon source is glucose, xylose, orglycerol.

In one embodiment, the fermentable carbon source is a mixture oflignin-derived aromatic monomers and/or oligomers.

In one embodiment, the fermentable carbon source is biomass hydrolysate.

Host Cells

The production organisms (e.g., a recombinant host cell) may include anyorganism capable of expressing the genes required for the production ofa biochemical product of interest, such as phenol, catechol or muconicacid (e.g., microorganism or plant). For example, the productionorganism may be a microorganism or plant. Microorganisms include, butare not limited to enteric bacteria (Escherichia and Salmonella, forexample) as well as Bacillus, Sphingomonas, Clostridium, Acinetobacter,Actinomycetes such as Streptomyces, Corynebacterium; methanotrophs suchas Methylosinus, Methylomonas, Rhodococcus and Pseudomonas;cyanobacteria, such as Synechococcus and Synechocystis; yeasts, such asSaccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula,Debaryomyces, Mucor, Pichia, Yarrowia, and Torulopsis; filamentousfungi, such as Aspergillus and Arthrobotrys; and algae, such asChlamydomonas, for example.

Accordingly, in one embodiment, the recombinant host cell is selectedfrom the group consisting of bacteria, yeast, filamentous fungi,cyanobacteria, algae, and plant cells.

In one embodiment, the recombinant host cell is selected from the groupconsisting of Escherichia, Salmonella, Bacillus, Acinetobacter,Streptomyces, Sphingomonas, Clostridium, Corynebacterium, Methylosinus,Methylomonas, Rhodococcus, Pseudomonas, Rhodobacter, Synechocystis,Saccharomyces, Klebsiella, Zygosaccharomyces, Kluyveromyces, Candida,Hansenula, Debaryomyces, Mucor, Pichia, Yarrowia, Torulopsis,Aspergillus, Arthrobotrys, Brevibacterium, Microbacterium, Arthrobacter,Ctirobacter, Chlamydomonas, and Zymomonas.

In certain embodiments, the recombinant host cell is Escherichia coli.

In certain embodiments, the recombinant host cell is E. coli NST74, E.coli NST74 ΔpheA, E. coli NST74 ΔpheA ΔpykA ΔpykF, or E. coli NST74ΔpheA ΔpykA ΔpykF Δcrr.

In certain embodiments, the recombinant host cell is a cell as describedherein, such as in the Examples, Tables, or Figures.

Microbial expression systems and expression vectors containingregulatory sequences that direct high-level expression of foreignproteins and over-expression of native proteins are well known to thoseskilled in the art (see, e.g., the Example). Any of these could be usedfor recombinant expression of at least one gene described herein for theproduction of a biochemical product, including those as described herein(e.g., phenol, catechol or muconic acid). Such an expression vector(s)comprising the gene(s) of interest could then be introduced intoappropriate microorganisms via transformation to allow for expression ofthe enzyme(s).

Genes

Described herein are methods for the microbial production of biochemicalproducts, such as phenol, catechol, and muconic acid, from renewablesubstrates using recombinant host cells. Specifically, embodimentsdescribed herein may involve the incorporation of genes encodingpolypeptides having isochorismate synthase activity, isochorismatepyruvate lyase activity, salicylate decarboxylase activity, phenol2-monooxygenase activity, catechol-1,2-dioxygenase activity, tyrosinephenol lyase activity, chorismate lyase activity, chorismate pyruvatelyase activity, p-hydroxybenzoate decarboxylase activity,p-hydroxybenzoate hydroxylase activity, protocatechuate decarboxylaseactivity, and/or 3-dehydroshikimate dehydratase activity into a singlehost organism and the use of those organisms to convert renewableresources such as glucose, for example, to phenol, catechol and muconicacid. As discussed below, genes encoding enzymes having such activitiesare known in the art. In certain embodiments, a gene encoding apolypeptide having the specific activity described below is derived froman organism described herein.

Genes encoding a polypeptide having isochorismate synthase activity areknown in the art and several have been sequenced from both microbial andplant origin. The sequence of isochorismate synthase activity encodinggenes are available (for example, entC, menF, pchA, ICS1; see GenBankGene ID: 945511, 946712, 881821, and 843810). Accordingly, in certainembodiments, the gene encoding a polypeptide having isochorismatesynthase activity is entC, menF, pchA or ICS/. In certain embodiments,the gene encoding a polypeptide having isochorismate synthase activityis entC. In certain embodiments, the entC gene has the followingsequence:

(SEQ ID NO: 1) ATGGATACGTCACTGGCTGAGGAAGTACAGCAGACCATGGCAACACTTGCGCCCAATCGCTTTTTCTTTATGTCGCCGTACCGCAGTTTTACGACGTCAGGATGTTTCGCCCGCTTCGATGAACCGGCTGTGAACGGGGATTCGCCCGACAGTCCCTTCCAGCAAAAACTCGCCGCGCTGTTTGCCGATGCCAAAGCGCAGGGCATCAAAAATCCGGTGATGGTCGGGGCGATTCCCTTCGATCCACGTCAGCCTTCGTCGCTGTATATTCCTGAATCCTGGCAGTCGTTCTCCCGTCAGGAAAAACAAGCTTCCGCACGCCGTTTCACCCGCAGCCAGTCGCTGAATGTGGTGGAACGCCAGGCAATTCCGGAGCAAACCACGTTTGAACAGATGGTTGCCCGCGCCGCCGCACTTACCGCCACGCCGCAGGTCGACAAAGTGGTGTTGTCACGGTTGATTGATATCACCACTGACGCCGCCATTGATAGTGGCGTATTGCTGGAACGGTTGATTGCGCAAAACCCGGTTAGTTACAACTTCCATGTTCCGCTGGCTGATGGTGGCGTCCTGCTGGGGGCCAGCCCGGAACTGCTGCTACGTAAAGACGGCGAGCGTTTTAGCTCCATTCCGTTAGCCGGTTCCGCGCGTCGTCAGCCGGATGAAGTGCTCGATCGCGAAGCAGGTAATCGTCTGCTGGCGTCAGAAAAAGATCGCCATGAACATGAACTGGTGACTCAGGCGATGAAAGAGGTACTGCGCGAACGCAGTAGTGAGTTACACGTTCCTTCTTCTCCACAGCTGATCACCACGCCGACGCTGTGGCATCTCGCAACTCCCTTTGAAGGTAAAGCGAATTCGCAAGAAAACGCACTGACTCTGGCCTGTCTGCTGCATCCGACCCCCGCGCTGAGCGGTTTCCCGCATCAGGCCGCGACCCAGGTTATTGCTGAACTGGAACCGTTCGACCGCGAACTGTTTGGCGGCATTGTGGGTTGGTGTGACAGCGAAGGTAACGGCGAATGGGTGGTGACCATCCGCTGCGCGAAGCTGCGGGAAAATCAGGTGCGTCTGTTTGCCGGAGCGGGGATTGTGCCTGCGTCGTCACCGTTGGGTGAGTGGCGCGAAACAGGCGTCAAACTTTCTACCATGTTGAACGTTTTTGGATTGCATTAA.Accordingly, in certain embodiments, the at least one gene encoding apolypeptide having isochorismate synthase activity comprises/consists ofa sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQID NO:1.

Genes encoding polypeptides having isochorismate pyruvate lyase activityare known in the art and several have been sequenced from microbialorigin. The sequence of isochorismate pyruvate lyase encoding genes areavailable (for example, pchB; see GenBank Gene ID: 881846). Accordingly,in certain embodiments, the gene encoding a polypeptide havingisochorismate pyruvate lyase activity is pchB. In certain embodiments,the pchB gene has the following sequence:ATGAAAACTCCCGAAGACTGCACCGGCCTGGCGGACATCCGCGAGGCCATCGACCGGATCGACCTGGATATCGTCCAGGCCCTCGGCCGCCGCATGGACTACGTCAAGGCGGCGTCGCGCTTCAAGGCCAGCGAGGCGGCGATTCCGGCGCCCGAGCGGGTCGCCGCGATGCTCCCCGAGCGCGCCCGCTGGGCCGAGGAAAACGGACTCGACGCGCCCTTCGTCGAGGGACTGTTCGCGCAGATCATCCACTGGTACATCGCCGAGCAGATCAAGTACTGGCGCCAGACACGGGGTGCCGCATGA (SEQ ID NO:2). Accordingly, in certainembodiments, the at least one gene encoding a polypeptide havingisochorismate pyruvate lyase activity comprises/consists of a sequencehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2.

Genes encoding polypeptides having salicylate decarboxylase activity areknown in the art and to date only one has been sequenced from microbialorigin. The sequence of salicylate decarboxylase encoding genes areavailable (for example, SDC; see DDBJ ID: DM040453). Accordingly, incertain embodiments, the gene encoding a polypeptide having salicylatedecarboxylase activity is SDC. In certain embodiments, the SDC gene hasthe following sequence:ATGCGTGGTAAAGTTAGCCTGGAAGAAGCATTTGAACTGCCGAAATTTGCAGCACAGACCAAAGAAAAAGCCGAACTGTATATTGCACCGAATAATCGCGATCGCTATTTTGAAGAAATTCTGAATCCGTGTGGTAATCGTCTGGAACTGAGCAATAAACATGGTATTGGCTATACCATCTATAGCATCTATTCACCGGGTCCGCAGGGTTGGACCGAACGTGCAGAATGTGAAGAATATGCACGTGAATGCAACGATTATATCAGCGGTGAAATTGCCAATCACAAAGATCGTATGGGTGCATTTGCAGCCCTGAGCATGCATGATCCGAAACAGGCAAGCGAAGAACTGACCCGTTGTGTTAAAGAACTGGGTTTTCTGGGTGCACTGGTTAATGATGTTCAGCATGCAGGTCCGGAAGGTGAAACCCATATCTTTTATGATCAGCCGGAATGGGATATCTTTTGGCAGACCTGTGTTGATCTGGATGTTCCGTTTTATCTGCATCCGGAACCGCCTTTTGGTAGCTATCTGCGTAATCAGTATGAAGGTCGCAAATATCTGATTGGTCCGCCTGTTAGCTTTGCAAATGGTGTTAGCCTGCATGTTCTGGGTATGATTGTTAATGGTGTGTTTGATCGTTTTCCGAAACTGAAAGTTATTCTGGGTCATCTGGGTGAACATATTCCGGGTGATTTTTGGCGTATTGAACATTGGTTTGAACACTGTAGCCGTCCGCTGGCAAAAAGCCGTGGTGATGTTTTTGCAGAAAAACCGCTGCTGCATTATTTTCGCAATAACATTTGGCTGACCACGAGCGGCAATTTTAGCACCGAAACCCTGAAATTTTGCGTTGAACATGTTGGTGCAGAACGCATTCTGTTTAGCGTTGATAGCCCGTATGAACATATCGATGTTGGTTGTGGTTGGTATGATGATAATGCCAAAGCAATTATGGAAGCCGTTGTGGTGAAAAAGCCTATAAAGATATTGGTCGCGACAACGCGAAAAAACTGTTTAAACTGGGCAAATTCTATGACAGCGAAGCCTAA (SEQ ID NO:3). Accordingly, in certainembodiments, the at least one gene encoding a polypeptide havingsalicylate decarboxylase activity comprises/consists of a sequencehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:3.

Genes encoding polypeptides having phenol 2-monooxygenase activity areknown in the art and have been sequenced. The sequence of phenol2-monooxygenase encoding genes are available (for example, operondmpLMNOP, loci GI: 170525 and operon phKLMNOP; see GenBankAccession:L04488.1, M60276.1). Accordingly, in certain embodiments, the geneencoding a polypeptide having phenol 2-monooxygenase activity is operondmpLMNOP, loci GI: 170525 or operon phKLMNOP, loci GI: 151449. Incertain embodiments, the genes encoding a polypeptide having phenol2-monooxygenase activity is operon phKLMNOP. In certain embodiments, thephKLMNOP operon has the following sequence:GAGCTCGTGCTGCCTCACGAGGCCTTCGAGATTTTCTGCAAACATAACAAAGTCGTCCACATGGACTCCAACATAATCCGCAAAATTGACGAAGACATGGTCAAGTGGCGGTTCGGAGAGCATGGCAAGCGCTACTGAGCAAGACGCATCGGCATACAGTATCACAACAACAACACGGTAAGGTTGATATGAGTATTGAAATCAAGACCAATTCGGTGGAACCTATCCGCCATACTTATGGCCACATCGCCCGTCGCTTCGGTGATAAGCCGGCTACCCGTTATCAGGAGGCCAGCTACGACATTGAGGCAAAGACCAATTTCCATTACCGGCCCCAGTGGGATTCCGAGCACACCCTGAACGATCCCACGCGTACCGCCATCCGCATGGAAGACTGGTGCGCCGTTTCCGATCCCCGCCAGTTTTACTATGGCGCCTATGTCGGCAACCGGGCCAAGATGCAGGAGTCGGCCGAGACCAGCTTTGGCTTCTGCGAAAAGCGTAATCTGCTGACCCGCCTTTCCGAAGAAACCCAGAAGCAATTGTTGCGGCTGCTGGTGCCCCTGCGTCATGTCGAGCTTGGCGCCAACATGAACAACGCCAAGATCGCCGGTGATGCCACCGCCACGACCGTCTCCCAGATGCACATCTACACTGGGATGGATCGCTTGGGCATTGGCCAGTACCTGTCCCGTATTGCATTGATGATTGATGGCAGCACCGGTGCCGCTCTGGATGAGTCCAAGGCCTACTGGATGGATGACGAAATGTGGCAACCCATGCGCAAGCTGGTCGAAGACACGCTTGTGGTCGATGATTGGTTTGAGCTGACTCTGGTTCAGAACATTCTTATCGACGGAATGATGTACCCGCTGGTCTACGACAAGATGGACCAGTGGTTCGAAAGCCAGGGTGCTGAAGATGTGTCCATGCTCACGGAGTTCATGCGTGACTGGTACAAGGAATCCCTACGCTGGACTAATGCCATGATGAAAGCCGTGGCCGGTGAAAGTGAGACTAACCGTGAGTTGCTTCAAAAATGGATCGATCACTGGGAACCGCAGGCCTACGAAGCCCTGAAACCTCTGGCCGAAGCCTCCGTTGGCATCGACGGGCTGAATGAAGCCCGGGCGGAACTCTCTGCCCGCCTGAAGAAATTCGAACTGCAGAGCCGGGGAGTCTCAGCATGAGCCAGCTTGTATTTATTGTATTCCAGGACAACGACGACTCCCGCTACCTCGCGGAAGCCGTTATGGAAGATAACCCCGACGCCGAAATGCAGCACCAGCCGGCCATGATCCGGATCCAGGCGGAAAAACGTCTGGTGATCAACCGCGAAACCATGGAAGAAAAGCTGGGGCGAGACTGGGATGTTCAGGAAATGCTCATAAATGTTATCAGCATCGCCGGCAACGTCGATGAAGACGATGATCACTTCATTCTTGAATGGAATTAATCGGGAGAAACATCATGGTTAGTAAAAACAAAAAGCTTAACCTTAAAGACAAGTATCAATACCTGACCCGGGATATGGCCTGGGAACCGACCTATCAGGACAAGAAAGATATTTTTCCGGAGGAGGATTTTGAGGGTATCAAGATCACCGACTGGTCCCAGTGGGAAGATCCGTTCCGCCTGACCATGGATGCCTACTGGAAATACCAGGCGGAAAAAGAGAAGAAGCTGTACGCCATTTTCGATGCATTTGCCCAGAACAACGGCCACCAGAACATTTCAGACGCCCGTTATGTGAACGCGCTAAAACTGTTCATCAGTGGTATATCTCCGCTTGAACATGCGGCGTTCCAGGGTTATTCCAAGGTCGGTCGCCAGTTTAGCGGCGCCGGGGCGCGGGTTGCCTGCCAGATGCAGGCAATTGACGAGCTGCGTCATTCCCAGACCCAGCAACACGCGATGAGCCACTACAACAAGCACTTCAACGGTCTGCACGATGGCCCGCACATGCACGATCGGGTGTGGTACCTGTCGGTGCCGAAATCGTTCTTTGATGATGCACGCTCGGCTGGTCCGTTCGAGTTCCTGACGGCCATCTCATTCTCGTTCGAGTATGTGCTCACCAACCTGTTGTTCGTACCGTTCATGTCGGGCGCTGCCTATAACGGCGACATGGCGACAGTCACCTTCGGTTTCTCCGCCCAGTCTGACGAAGCCCGTCATATGACCCTGGGCCTTGAGGTGATCAAGTTCATCCTCGAGCAGCACGAAGATAACGTGCCCATCGTTCAGCGCTGGATCGACAAGTGGTTCTGGCGCGGATTTCGCCTGCTTAGCCTGGTCAGCATGATGATGGACTACATGCTGCCAAACAAGGTCATGTCCTGGTCCGAGGCATGGGAAGTCTATTACGAGCAGAACGGCGGTGCTCTGTTCAAGGACCTGGAGCGATACGGCATCCGCCCGCCCAAATACCAGGACGTGGCTAACGATGCCAAACATCACCTGAGCCACCAGCTTTGGACCACTTTCTACCAGTACTGCCAGGCCACCAACTTCCATACTTGGATTCCGGAGAAGGAAGAGATGGACTGGATGTCCGAGAAGTATCCGGACACTTTCGACAAGTACTACCGTCCGCGTTACGAGTACCTGGCGAAAGAGGCTGCCGCTGGCCGTCGCTTCTACAACAACACCCTGCCGCAGCTGTGCCAAGTGTGTCAGATCCCGACCATTTTCACCGAGAAAGATGC CCCAACCATGCTCAGCCATCGGCAGATAGAACATGAGGGCGAACGCTATCACTTCTGCTCTGACGGCTGCTGCGACATCTTCAAACACGAGCCGGAGAAGTACATACAGGCCTGGCTGCCGGTGCACCAGATCTACCAGGGCAACTGTGAAGGCGGGGATCTCGAGACCGTGGTGCAGAAGTATTACCACATCAATATCGGAGAGGACAATTTCGACTACGTTGGATCGCCCGACCAGAAACACTGGCTGTCGATCAAGGGCCGGAAGCCTGCAGACAAGAACCAGGACGCCGCCTGATATTGATTGGAGAGTCGCCCGGTAGCCGCTGGCACCGGGTGAAACACCCATAAAAACAACGAGGTGACCATCATGAGTGTAAACGCACTTTACGACTACAAGTTTGAACCTAAAGACAAGGTCGAGAACTTCCACGGCATGCAGCTGCTGTATGTCTACTGGCCCGATCACCTGCTGTTCTGCGCGCCCTTCGCGCTGCTGGTGCAGCCGGGTATGACCTTCAGTGCCCTGGTGGACGAGATTCTCAAGCCGGCTACCGCCGCGCACCCGGACTCTGCCAAGGCGGACTTCCTGAATGCCGAGTGGTTGCTGAACGATGAACCGTTCACACCCAAGGCTGACGCCAGCCTGAAAGAGCAGGGTATTGATCACAAGAGCATGCTGACGGTGACCACGCCGGGCCTGAAGGGCATGGCGAACGCCGGTTACTGAGGGTAGCACTATGAGTTACACCGTCACTATTGAGCCGATCGGCGAGCAGATTGAGGTAGAGGATGGCCAGACTATCCTCGCCGCCGCCCTGCGCCAGGGTGTCTGGCTGCCCTTTGCCTGCGGCCACGGCACCTGTGCTACCTGTAAGGTTCAGGTGCTTGAAGGTGATGTCGAGATCGGAAACGCCTCGCCCTTTGCGCTGATGGATATCGAACGTGACGAGGGCAAGGTTCTGGCCTGCTGCGCCACGGTTGAGAGCGACGTCACCATTGAGGTGGACATCGATGTGGATCCGGATTTTGAGGGCTACCCGGTGGAGGACTATGCCGCCATAGCGACCGATATCGTCGAACTCTCTCCGACCATCAAGGGCATTCACCTGAAACTGGACCGGCCGATGACATTCCAGGCCGGCCAGTACATCAATATCGAACTGCCGGGTGTTGAAGGCGCGAGGGCCTTCTCCCTGGCCAACCCGCCCAGCAAAGCAGACGAAGTGGAGCTGCATGTGCGCCTCGTTGAGGGCGGTGCTGCCACCACCTACATCCACGAACAACTGAAAACGGGTGATGCGCTGAACCTTTCAGGCCCTTACGGCCAGTTCTTCGTGCGTAGTTCCCAACCCGGCGATCTGATTTTCATCGCCGGCGGATCCGGATTGTCCAGTCCCCAGTCGATGATCCTTGATCTGCTTGAGCAGAACGATGAGCGCAAGATCGTTCTGTTCCAGGGTGCCCGAAACCTGGCAGAGCTTTACAACCGGGAGCTGTTTGAGGCTCTGGATCGCGACCACGACAATTTCACCTACGTACCGGCGCTTAGCCAAGCCGACGAAGACCCTGACTGGAAGGGCTTCCGAGGCTATGTCCATGAGGCGGCCAACGCCCATTTCGATGGCCGGTTTGCCGGTAACAAGGCATACCTGTGCGGCCCGCCTCCAATGATCGATGCGGCTATCACGGCATTGATGCAGGGGCGGCTGTTCGAGCGTGACATCTTCATGGAGAAATTCCTGACAGCGGCGGACGGAGCTGAAGACACCCAGCGTTCGGCCCTGTTCAAGAAGATATA G (SEQ IDNO:4). Accordingly, in certain embodiments, the at least one geneencoding a polypeptide having phenol 2-monooxygenase activitycomprises/consists of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:4.

Genes encoding polypeptides having catechol-1,2-dioxygenase activity areknown in the art and have been sequenced. The sequence ofcatechol-1,2-dioxygenase encoding genes are available (for example,catA, salD; see, GenBank Gene ID: 3609645, 3614680, 879147, 5191661 and5191980). Accordingly, in certain embodiments, the gene encoding apolypeptide having catechol-1,2-dioxygenase activity is catA, or salD.In certain embodiments, the gene encoding a polypeptide havingcatechol-1,2-dioxygenase activity is catA. In certain embodiments, thecatA gene has the following sequence:ATGACCGTGAAAATTTCCCACACTGCCGACATTCAAGCCTTCTTCAACCGGGTAGCTGGCCTGGACCATGCCGAAGGAAACCCGCGCTTCAAGCAGATCATTCTGCGCGTGCTGCAAGACACCGCCCGCCTGATCGAAGACCTGGAGATTACCGAGGACGAGTTCTGGCACGCCGTCGACTACCTCAACCGCCTGGGCGGCCGTAACGAGGCAGGCCTGCTGGCTGCTGGCCTGGGTATCGAGCACTTCCTCGACCTGCTGCAGGATGCCAAGGATGCCGAAGCCGGCCTTGGCGGCGGCACCCCGCGCACCATCGAAGGCCCGTTGTACGTTGCCGGGGCGCCGCTGGCCCAGGGCGAAGCGCGCATGGACGACGGCACTGACCCAGGCGTGGTGATGTTCCTTCAGGGCCAGGTGTTCGATGCCGACGGCAAGCCGTTGGCCGGTGCCACCGTCGACCTGTGGCACGCCAATACCCAGGGCACCTATTCGTACTTCGATTCGACCCAGTCCGAGTTCAACCTGCGTCGGCGTATCATCACCGATGCCGAGGGCCGCTACCGCGCGCGCTCGATCGTGCCGTCCGGGTATGGCTGCGACCCGCAGGGCCCAACCCAGGAATGCCTGGACCTGCTCGGCCGCCACGGCCAGCGCCCGGCGCACGTGCACTTCTTCATCTCGGCACCGGGGCACCGCCACCTGACCACGCAGATCAACTTTGCTGGCGACAAGTACCTGTGGGACGACTTTGCCTATGCCACCCGCGACGGGCTGATCGGCGAACTGCGTTTTGTCGAGGATGCGGCGGCGGCGCGCGACCGCGGTGTGCAAGGCGAGCGCTTTGCCGAGCTGTCATTCGACTTCCGCTTGCAGGGTGCCAAGTCGCCTGACGCCGAGGCGCGAAGCCATCGGCCGCGGGCGTTGCAGGAGGGCTGA (SEQ ID NO:5). Accordingly, incertain embodiments, the at least one gene encoding a polypeptide havingcatechol-1,2-dioxygenase activity comprises/consists of a sequencehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5.

Genes encoding polypeptides having tyrosine phenol lyase activity areknown in the art and have been sequenced. The sequence of tyrosinephenol lyase encoding genes are available (for example, tutA; seeGenBank Gene ID: L08484.1). Accordingly, in certain embodiments, thegene encoding a polypeptide having tyrosine phenol lyase activity istutA. In certain embodiments, the tutA gene has the following sequence:ATGAATTATCCGGCAGAACCCTTCCGTATTAAAAGCGTTGAAACTGTATCTATGATCCCGCGTGATGAACGCCTCAAGAAAATGCAGGAAGCGGGTTACAATACTTTCCTGTTAAATTCGAAAGATATTTATATTGACCTGCTGACAGACAGTGGCACTAACGCAATGAGCGACAAGCAGTGGGCCGGAATGATGATGGGTGATGAAGCGTACGCGGGCAGCGAAAACTTCTATCATCTGGAAAGAACCGTGCAGGAACTGTTCGGCTTTAAACATATTGTTCCGACTCACCAGGGGCGTGGCGCAGAAAACCTGTTATCGCAGTTGGCTATTAAACCTGGGCAATATGTTGCCGGGAATATGTATTTCACCACCACCCGTTATCACCAGGAAAAAAATGGTGCGGTGTTTGTCGATATCGTTCGTGACGAAGCGCACGATGCCGGTCTGAATATTGCGTTTAAAGGTGATATCGATCTTAAAAAATTACAAAAGCTGATTGATGAAAAAGGCGCAGAAAATATTGCGTATATCTGCCTGGCGGTGACGGTTAACCTCGCAGGTGGGCAGCCGGTCTCGATGGCCAACATGCGTGCGGTGCGTGAACTGACAGAAGCGCACGGCATTAAAGTGTTCTACGACGCCACCCGTTGCGTGGAAAACGCCTACTTTATCAAAGAGCAAGAGCAGGGCTTTGAGAACAAGAGCATCGCCGAGATCGTGCATGAGATGTTCAGCTACGCCGACGGTTGTACCATGAGTGGTAAAAAAGACTGTCTGGTGAACATCGGCGGTTTCCTGTGCATGAACGATGACGAAATGTTCTCTTCTGCCAAAGAGTTAGTCGTGGTCTACGAAGGGATGCCATCTTACGGCGGCCTGGCCGGACGTGATATGGAAGCCATGGCGATTGGCCTGCGCGAAGCCATGCAATACGAATATATTGAGCACCGCGTGAAGCAGGTTCGCTACCTGGGCGATAAGCTGAAAGCCGCTGGCGTACCGATTGTTGAACCGGTAGGCGGTCACGCGGTATTCCTCGATGCGCGTCGCTTCTGCGAGCATCTGACGCAGGACGAGTTCCCGGCGCAAAGCCTGGCGGCGAGCATTTATGTGGAAACTGGTGTGCGCAGTATGGAACGCGGAATAATCTCTGCAGGCCGTAATAACGTGACCGGTGAACACCACAGACCGAAACTGGAAACCGTGCGTCTGACTATTCCACGCCGCGTTTATACCTACGCGCACATGGATGTCGTAGCTGACGGTATTATTAAACTTTACCAGCACAAAGAAGATATTCGCGGGCTGAAGTTTATTTACGAGCCGAAGCAGTTGCGTTTCTTTACTGCACGCTTTGACTATATCTAA (SEQ ID NO:6). Accordingly, in certain embodiments,the at least one gene encoding a polypeptide having tyrosine phenollyase activity comprises/consists of a sequence having at least about50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to SEQ ID NO:6.

Genes encoding polypeptides having chorismate lyase activity are knownin the art and have been sequenced. The sequence of chorismate lyaseencoding genes are available (for example, ubiC; see GenBank Gene ID:ECK4031). Accordingly, in certain embodiments, the gene encoding apolypeptide having chorismate lyase activity is ubiC. In certainembodiments, the ubiC gene has the following sequence:

(SEQ ID NO: 7) ATGTCACACCCCGCGTTAACGCAACTGCGTGCGCTGCGCTATTGTAAAGAGATCCCTGCCCTGGATCCGCAACTGCTCGACTGGCTGTTGCTGGAGGATTCCATGACAAAACGTTTTGAACAGCAGGGAAAAACGGTAAGCGTGACGATGATCCGCGAAGGGTTTGTCGAGCAGAATGAAATCCCCGAAGAACTGCCGCTGCTGCCGAAAGAGTCTCGTTACTGGTTACGTGAAATTTTGTTATGTGCCGATGGTGAACCGTGGCTTGCCGGTCGTACCGTCGTTCCTGTGTCAACGTTAAGCGGGCCGGAGCTGGCGTTACAAAAATTGGGTAAAACGCCGTTAGGACGCTATCTGTTCACATCATCGACATTAACCCGGGACTTTATTGAGATAGGCCGTGATGCCGGGCTGTGGGGGCGACGTTCCCGCCTGCGATTAAGCGGTAAACCGCTGTTGCTAACAGAACTGTTTTTACCGGCGTCACCGTTGTACTAA.Accordingly, in certain embodiments, the at least one gene encoding apolypeptide having chorismate lyase activity comprises/consists of asequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQID NO:7.

Genes encoding polypeptides having p-hydroxybenzoate decarboxylaseactivity are known in the art and have been sequenced. The sequence ofp-hydroxybenzoate decarboxylase encoding genes are available (forexample, the operon kpdBCD; see GenBank Gene ID: KUP82937.1).Accordingly, in certain embodiments, the genes encoding a polypeptidehaving p-hydroxybenzoate decarboxylase activity is the kpdBCD operon. Incertain embodiments, the kpdBCD operon has the following sequence:ATGGGCCAAAACCACCATCGAGCTGGAAACGCCCTGGACAGCGCGCGAAGTGGCCGCGCTGGCGGACTTTTCCCACAGCCCGGCAGACCAGGCCGCCACCATCTCTTCCGGTTCATTTCGTACCGACGGCATGATCGTTATTCCCTGCAGTATGAAAACGCTGGCAGGCATTCGCGCGGGTTATGCCGAAGGGCTGGTGGGCCGCGCGGCGGACGTGGTGCTCAAAGAGGGGCGCAAGCTGGTGCTGGTCCCGCGGGAAATGCCGCTCAGCACGATCCATCTGGAGAACATGCTGGCGCTGTCGCGCATGGGCGTGGCGATGGTGCCGCCGATGCCGGCTTACTACAACCACCCGGAGACGGTTGACGATATCACCAATCATATCGTCACCCGGGTGCTGGATCAGTTTGGCCTCGACTATCACAAAGCGCGCCGCTGGAACGGCTTGCGCACGGCAGAACAATTTGCACAGGAGATCGAATAATGGCTTTTGATGATTTGCGCAGCTTTTTGCAGGCGCTGGATGACCAGGGACAACTGCTGAAAATCAGTGAAGAGGTGAACGCTGAGCCCGATCTGGCGGCGGCCGCCAATGCGACCGGACGCATCGGCGACGGCGCCCCGGCGCTGTGGTTCGATAATATTCGCGGCTTTACCGACGCCCGCGTGACGATGAACACCATCGGCTCGTGGCAGAACCATGCCATCTCGCTGGGCCTGCCGCCTAACACGCCGGTGAAAAAGCAGATTGATGAATTCATTCGCCGCTGGGATAACTTCCCGGTGACGCCAGAGCGCCGCGCCAACCCGGCGTGGGCGGAAAACACCGTGGATGGCGACGATATCAACCTGTTCGATATTCTGCCACTGTTCCGCCTCAACGATGGTGACGGCGGTTTCTACCTCGATAAAGCCTGTGTCGTATCACGCGACCCGCTTGATCCTGACAACTTCGGTAAGCAAAACGTCGGTATCTACCGCATGGAAGTGAAAGGCAAGCGCAAGCTCGGCCTGCAGCCGGTGCCGATGCACGATATCGCGCTGCATCTGCACAAAGCGGAAGAGCGTGGGGAAGATCTGCCGATCGCTATTACCCTCGGTAACGACCCGATTATTACCCTGATGGGCGCCACGCCGCTGAAATACGATCAATCAGAATATGAAATGGCTGGCGCGCTGCGCGAGAGCCCGTATCCCATCGCCACCGCGCCGCTGACCGGCTTTGACGTGCCCTGGGGTTCGGAAGTGATCCTCGAAGGGGTCATTGAAGGGCGTAAGCGTGAGATCGAGGGGCCGTTCGGTGAGTTTACCGGTCACTACTCCGGCGGTCGTAACATGACGGTAGTGCGTATCGACAAAGTCTCGTATCGCAGCAAACCGATTTTTGAATCGCTCTATCTCGGTATGCCGTGGACCGAGATTGACTATCTGATGGGCCCGGCGACCTGCGTGCCGCTGTATCAGCAGTTGAAGGCAGAGTTCCCGGAAGTGCAGGCGGTCAACGCCATGTACACCCATGGTCTGCTGGCGATCATCTCCACCAAAAAACGCTACGGCGGTTTTGCCCGCGCGGTGGGCCTGCGGGCGATGACCACTCCGCACGGCCTCGGCTATGTGAAGATGGTGATCATGGTTGATGAAGACGTCGACCCGTTCAACCTGCCGCAGGTGATGTGGGCGCTCTCCTCGAAAGTTAACCCGGCGGGTGACCTGGTGCAGTTGCCGAACATGTCGGTCCTTGAACTTGACCCTGGCTCCAGCCCGGCAGGCATCACCGACAAACTGATTATCGACGCCACCACCCCGGTTGCGCCGGACCTTCGCGGCCACTACAGCCAGCCGGTGCAGGATCTGCCGGAAACCAAAGCCTGGGCTGAAAAACTGACCGCTATGCTGGCCAACCGTAAATAAGGAGAAGAAGATGATTTGTCCACGTTGCGCCGATGAAAAGATTGAAGTGATGGCAACCTCGCCGGTGAAAGGGGTCTGGACCGTGTATCAGTGCCAGCACTGTCTTTACACCTGGCGAGATACCGAGCCGCTGCGCCGCACCAGTCGCGAACACTATCCGGAAGCGTTCCGCATGACGCAGAAAGATATTGATGAGGCACCGCAGGTGCCACACGTACCGCCGCTATTGCCGGA AGATAAGCGTTAA(SEQ ID NO:8). Accordingly, in certain embodiments, the at least onegene encoding a polypeptide having p-hydroxybenzoate decarboxylaseactivity comprises/consists of a sequence having at least about 50%,55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to SEQ ID NO:8.

Genes encoding polypeptides having p-hydroxybenzoate hydroxylaseactivity are known in the art and have been sequenced. The sequence ofp-hydroxybenzoate hydroxylase encoding genes are available (for example,pobA; see GenBank Gene ID: AAA88455.1). Accordingly, in certainembodiments, the gene encoding a polypeptide having p-hydroxybenzoatehydroxylase activity is pobA. In certain embodiments, the pobA gene hasthe following sequence:ATGAAGACTCAAGTCGCCATCATCGGCGCCGGTCCGTCCGGCCTCCTGCTCGGCCAGTTGCTGCACAAGGCCGGCATCGACAACGTGATCCTCGAACGCCAGACCCCGGACTACGTGCTCGGCCGCATCCGCGCCGGCGTGCTGGAACAGGGTATGGTCGACCTGCTGCGCGAGGCCGGCGTCGACCGGCGCATGGCGCGCGACGGGCTGGTCCACGAAGGCGTGGAGATCGCCTTCGCCGGGCAGCGCCGGCGCATCGACCTGAAGCGCCTGAGCGGCGGCAAGACGGTGACGGTCTACGGCCAGACCGAGGTCACCCGCGACCTCATGGAGGCCCGCGAAGCCTGCGGCGCCACTACCGTCTACCAGGCCGCCGAGGTGCGCCTGCACGACCTGCAAGGTGAGCGCCCCTACGTGACCTTCGAACGCGACGGCGAACGGCTGCGCCTGGATTGCGACTACATCGCCGGCTGCGATGGCTTCCACGGCATCTCGCGGCAATCGATCCCGGCGGAGCGGCTGAAGGTCTTCGAGCGGGTCTATCCGTTCGGCTGGCTCGGCCTGCTCGCCGACACCCCGCCGGTGAGCCACGAACTGATCTACGCCAACCATCCGCGCGGCTTCGCCCTGTGCAGCCAGCGTTCGGCCACCCGCAGCCGCTACTACGTGCAGGTGCCATTGTCGGAGAAGGTCGAGGACTGGTCCGACGAGCGCTTCTGGACGGAACTGAAGGCGCGACTCCCGTCCGAGGTGGCGGAGAAACTGGTGACCGGACCTTCGCTGGAGAAGAGCATCGCGCCGCTGCGCAGCTTCGTGGTCGAGCCGATGCAGCATGGCCGGCTGTTCCTCGCCGGCGACGCCGCGCACATCGTGCCGCCCACCGGCGCCAAGGGACTGAACCTGGCCGCCAGCGACGTCAGCACGCTCTACCGGCTGCTGCTGAAGGCCTACCGCGAAGGGCGCGGCGAACTGCTGGAACGCTATCGGCAATCTGCCTGCGGCGGATCTGGAAGGCCGAACGCTTCTCCTGGTGGATGACTTCGGTGCTGCATCGCTTCCCCGACACCGACGCGTTCAGCCAGCGCATCCAGCAGACCGAACTGGAGTATTACCTGGGCTCCGAGGCGGGCCTGGCGACCATCGCCGAGAACTATGTCGGCCTGCCCTACGAGGAAATCGA GTAG (SEQ IDNO:9). Accordingly, in certain embodiments, the at least one geneencoding a polypeptide having p-hydroxybenzoate hydroxylase activitycomprises/consists of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:9.

Genes encoding polypeptides having protocatechuate decarboxylaseactivity are known in the art and have been sequenced. The sequence ofprotocatechuate decarboxylase encoding genes are available (for example,aroY; see GenBank Gene ID: BAH20873.2.). Accordingly, in certainembodiments, the gene encoding a polypeptide having protocatechuatedecarboxylase activity is aroY. In certain embodiments, the aroY genehas the following sequence:

(SEQ ID NO: 10) ATGACCGCACCGATTCAGGATCTGCGCGACGCCATCGCGCTGCTGCAACAGCATGACAATCAGTATCTCGAAACCGATCATCCGGTTGACCCTAACGCCGAGCTGGCCGGTGTTTATCGCCATATCGGCGCGGGCGGCACCGTGAAGCGCCCCACCCGCATCGGGCCGGCGATGATGTTTAACAATATTAAGGGTTATCCACACTCGCGCATTCTGGTGGGTATGCACGCCAGCCGCCAGCGGGCCGCGCTGCTGCTGGGCTGCGAAGCCTCGCAGCTGGCCCTTGAAGTGGGTAAGGCGGTGAAAAAACCGGTCGCGCCGGTGGTCGTCCCGGCCAGCAGCGCCCCCTGCCAGGAACAGATCTTTCTGGCCGACGATCCGGATTTTGATTTGCGCACCCTGCTTCCGGCGCACACCAACACCCCTATCGACGCCGGCCCCTTCTTCTGCCTGGGCCTGGCGCTGGCCAGCGATCCCGTCGACGCCTCGCTGACCGACGTCACCATCCACCGCTTGTGCGTCCAGGGCCGGGATGAGCTGTCGATGTTTCTTGCCGCCGGCCGCCATATCGAAGTGTTTCGCCAAAAGGCCGAGGCCGCCGGCAAACCGCTGCCGATAACCATCAATATGGGTCTCGATCCGGCCATCTATATTGGCGCCTGCTTCGAAGCCCCTACCACGCCGTTCGGCTATAATGAGCTGGGCGTCGCCGGCGCGCTGCGTCAACGTCCGGTGGAGCTGGTTCAGGGCGTCAGCGTCCCGGAGAAAGCCATCGCCCGCGCCGAGATCGTTATCGAAGGTGAGCTGTTGCCTGGCGTGCGCGTCAGAGAGGATCAGCACACCAATAGCGGCCACGCGATGCCGGAATTTCCTGGCTACTGCGGCGGCGCTAATCCGTCGCTGCCGGTAATCAAAGTCAAAGCAGTGACCATGCGAAACAATGCGATTCTGCAGACCCTGGTGGGACCGGGGGAAGAGCATACCACCCTCGCCGGCCTGCCAACGGAAGCCAGTATCTGGAATGCCGTCGAGGCCGCCATTCCGGGCTTTTTACAAAATGTCTACGCCCACACCGCGGGTGGCGGTAAGTTCCTCGGGATCCTGCAGGTGAAAAAACGTCAACCCGCCGATGAAGGCCGGCAGGGGCAGGCCGCGCTGCTGGCGCTGGCGACCTATTCCGAGCTAAAAAATATTATTCTGGTTGATGAAGATGTCGACATCTTTGACAGCGACGATATCCTGTGGGCGATGACCACCCGCATGCAGGGGGACGTCAGCATTACGACAATCCCCGGCATTCGCGGTCACCAGCTGGATCCGTCCCAGACGCCGGAATACAGCCCGTCGATCCGTGGAAATGGCATCAGCTGCAAGACCATTTTTGACTGCACGGTCCCCTGGGCGCTGAAATCGCACTTTGAGCGCGCGCCGTTTGCCGACGTCGATCCGCGTCCGTTTGCACCGGAGTATTTCGCCCGGCTGGAAAAAAACCAGGGTAGC GCAAAATAA.Accordingly, in certain embodiments, the at least one gene encoding apolypeptide having protocatechuate decarboxylase activitycomprises/consists of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:10.

Genes encoding polypeptides having 3-dehydroshikimate dehydrataseactivity are known in the art and have been sequenced. The sequence of3-dehydroshikimate dehydratase encoding genes are available (forexample, aroZ, quiC, qsuB; see GenBank Gene IDs: 5987244, BAF53460.1).Accordingly, in certain embodiments, the gene encoding a polypeptidehaving 3-dehydroshikimate dehydratase activity is qsuB. In certainembodiments, the qsuB gene has the following sequence:

(SEQ ID NO: 11) ATGCGTACATCCATTGCCACTGTTTGTTTGTCCGGAACTCTTGCTGAAAAGCTGCGCGCAGCTGCAGATGCTGGATTTGATGGTGTGGAAATCTTCGAGCAGGACTTGGTGGTTTCCCCGCATTCGGCAGAGCAGATTCGTCAGCGGGCTCAGGATTTGGGATTAACCCTGGATCTGTTCCAGCCGTTTCGAGATTTCGAAGGTGTGGAAGAAGAGCAGTTTCTGAAGAATCTGCACCGCTTGGAAGAGAAGTTCAAGCTGATGAACAGGCTTGGCATTGAGATGATCTTGTTGTGTTCCAATGTGGGCACCGCGACCATCAATGATGATGACCTTTTCGTGGAGCAGTTGCATCGTGCAGCAGATTTGGCTGAGAAGTACAACGTCAAGATTGCTTATGAAGCGTTGGCGTGGGGCAAGTTTGTCAATGATTTTGAGCATGCGCATGCACTTGTGGAGAAGGTGAATCACAAGGCGCTGGGAACCTGCTTGGATACGTTCCATATTCTTTCCCGTGGTTGGGAAACCGACGAGGTGGAGAACATCCCTGCGGAGAAGATCTTCTTTGTTCAGTTAGCGGATGCGCCGAAGCTGAGCATGGACATTTTGTCCTGGTCGCGTCACCACCGTGTTTTCCCTGGTGAAGGCGATTTCGATCTGGTGAAATTCATGGTTCATCTGGCCAAGACGGGTTATGATGGCCCGATTTCTTTGGAGATCTTCAACGATTCCTTCCGCAAGGCCGAGGTTGGTCGCACCGCGATTGATGGGTTGCGTTCTTTGCGTTGGTTGGAAGATCAGACCTGGCATGCGCTAAATGCTGAGGATCGTCCAAGCGCTCTTGAACTGCGTGCACTTCCTGAGGTCGCGGAACCTGAGGGTGTTGATTTCATTGAGATCGCCACTGGACGTTTGGGTGAGACCATTCGGGTTCTTCATCAATTGGGTTTCCGCTTGGGTGGTCATCACTGCAGTAAGCAGGATTACCAGGTATGGACCCAGGGCGATGTGCGCATTGTGGTGTGTGATCGTGGGGTCACCGGGGCTCCAACCACGATCTCTGCGATGGGCTTTGACACCCCCGATCCAGAAGCTGCTCATGCCCGTGCGGAATTGCTGCGGGCTCAGACAATTGATCGTCCCCACATCGAGGGCGAAGTTGACCTAAAAGGTGTGTACGCACCGGATGGGGTGGAGCTGTTTTTCGCGGGGCCGAGCCCCGATGGAATGCCCGAGTGGCTGCCGGAATTCGGCGTCGAAAAGCAAGAAGCTGGTCTCATTGAAGCCATCGACCACGTCAATTTCGCCCAGCCGTGGCAACATTTTGATGAGGCAGTGCTGTTTTACACCGCGCTGATGGCGTTGGAGACTGTGCGTGAGGATGAGTTCCCGAGCCCAATTGGTTTGGTGCGCAATCAGGTGATGCGTTCGCCGAATGATGCGGTGCGGTTGCTGCTCAGCGTGGCGCCGGAGGACGGTGAGCAGGGAGATTTCCTCAACGCGGCCTACCCGGAGCACATTGCGTTGGCCACGGCGGACATCGTGGCGGTGGCTGAACGTGCGCGCAAACGAGGCCTGGATTTCTTGCCCGTCCCAGAGAATTACTACGACGATGTGCAGGCGCGTTTTGATTTGCCGCAGGAATTCTTGGACACACTCAAGGAAAACCACCTGCTTTACGACCGCGACGAGAACGGCGAATTCCTCCACTTTTACACCCGCACGTTGGGCACGCTGTTCTTCGAAGTGGTGGAACGCCGCGGCGGTTTTGCAGGTTGGGGCGAAACAAACGCTCCGGTGCGGTTGGCGGCGCAGTATCGTGAGGTGCGGGACCTCGAGCGGGGAATCCC AAACTAG.Accordingly, in certain embodiments, the at least one gene encoding apolypeptide having 3-dehydroshikimate dehydratase activitycomprises/consists of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:11.

Genes encoding polypeptides having feedback resistant (fbr) chorismatemutase/prephenate dehydrogenase activity are known in the art and havebeen sequenced. The sequence of chorismate mutase/prephenatedehydrogenase encoding genes are available (for example, tyrA^(fbr); seeGenBank Gene ID: AAA24331.1.). Accordingly, in certain embodiments, thegene encoding a polypeptide having feedback resistant chorismatemutase/prephenate dehydrogenase activity is tyrA^(fbr). In certainembodiments, the tyrA^(fbr) gene has the following sequence:ATGGTTGCTGAATTGACCGCATTACGCGATCAAATTGATGAAGTCGATAAAGCGCTGCTGAATTTATTAGCGAAGCGTCTGGAACTGGTTGCTGAAGTGGGCGAGGTGAAAAGCCGCTTTGGACTGCCTATTTATGTTCCGGAGCGCGAGGCATCTATGTTGGCCTCGCGTCGTGCAGAGGCGGAAGCTCTGGGTGTACCGCCGGATCTGATTGAGGATGTTTTGCGTCGGGTGATGCGTGAATCTTACTCCAGTGAAAACGACAAAGGATTTAAAACACTTTGTCCGTCACTGCGTCCGGTGGTTATCGTCGGCGGTGGCGGTCAGATGGGACGCCTGTTCGAGAAGATGCTGACCCTCTCGGGTTATCAGGTGCGGATTCTGGAGCAACATGACTGGGATCGAGCGGCTGATATTGTTGCCGATGCCGGAATGGTGATTGTTAGTGTGCCAATCCACGTTACTGAGCAAGTTATTGGCAAATTACCGCCTTTACCGAAAGATTGTATTCTGGTCGATCTGGCATCAGTGAAAAATGGGCCATTACAGGCCATGCTGGTGGCGCATGATGGTCCGGTGCTGGGGCTACACCCGATGTTCGGTCCGGACAGCGGTAGCCTGGCAAAGCAAGTTGTGGTCTGGTGTGATGGACGTAAACCGGAAGCATACCAATGGTTTCTGGAGCAAATTCAGGTCTGGGGCGCTCGGCTGCATCGTATTAGCGCCGTCGAGCACGATCAGAATATGGCGTTTATTCAGGCACTGCGCCACTTTGCTACTTTTGCTTACGGGCTGCACCTGGCAGAAGAAAATGTTCAGCTTGAGCAACTTCTGGCGCTCTCTTCGCCGATTTACCGCCTTGAGCTGGCGATGGTCGGGCGACTGTTTGCTCAGGACCCGCAGCTTTATGCCGACATCATTATGTCGTCAGAGCGTAATCTGGCGTTAATCAAACGTTACTATAAGCGTTTCGGCGAGGCGATTGAGTTGCTGGAGCAGGGCGATAAGCAGGCGTTTATTGACAGTTTCCGCAAGGTGGAGCACTGGTTCGGCGATTACGCACAGCGTTTTCAGAGTGAAAGCCGCGTGTTATTGCGTCAGGCGAATGACAATCGCCAGTAA (SEQ ID NO:56).Accordingly, in certain embodiments, the at least one gene encoding apolypeptide having chorismate mutase/prephenate dehydrogenase activitycomprises/consists of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:56.

Thus, in certain embodiments, the at least one gene encoding apolypeptide having isochorismate synthase activity comprises a sequencehaving at least about 70% sequence identity to SEQ ID NO:1; the at leastone gene encoding a polypeptide having isochorismate pyruvate lyaseactivity comprises a sequence having at least about 70% sequenceidentity to SEQ ID NO:2; the at least one gene encoding a polypeptidehaving salicylate decarboxylase activity comprises a sequence having atleast about 70% sequence identity to SEQ ID NO:3; the at least one geneencoding a polypeptide having phenol 2-monooxygenase activity comprisesa sequence having at least about 70% sequence identity to SEQ ID NO:4;the at least one gene encoding a polypeptide having 1,2-catecholdioxygenase activity comprises a sequence having at least about 70%sequence identity to SEQ ID NO:5; the at least one gene encoding apolypeptide having tyrosine phenol lyase activity comprises a sequencehaving at least about 70% sequence identity to SEQ ID NO:6; the at leastone gene encoding a polypeptide having chorismate lyase activitycomprises a sequence having at least about 70% sequence identity to SEQID NO:7; the at least one gene encoding a polypeptide havingp-hydroxybenzoate decarboxylase activity comprises a sequence having atleast about 70% sequence identity to SEQ ID NO:8; the at least one geneencoding a polypeptide having p-hydroxybenzoate hydroxylase activitycomprises a sequence having at least about 70% sequence identity to SEQID NO:9; the at least one gene encoding a polypeptide havingprotocatechuate decarboxylase activity comprises a sequence having atleast about 70% sequence identity to SEQ ID NO:10; and/or the at leastone gene encoding a polypeptide having 3-dehydroshikimate dehydrataseactivity comprises a sequence having at least about 70% sequenceidentity to SEQ ID NO:11.

In certain embodiments, the at least one gene encoding a polypeptidehaving isochorismate synthase activity is entC, menF, pchA or ICS1; theat least one gene encoding a polypeptide having isochorismate pyruvatelyase activity is pchB; the at least one gene encoding a polypeptidehaving salicylate decarboxylase activity is SDC; the at least one geneencoding a polypeptide having phenol 2-monooxygenase activity isdmpLMNOP or phKLMNOP; the at least one gene encoding a polypeptidehaving 1,2-catechol dioxygenase activity is catA or salD; the at leastone gene encoding a polypeptide having tyrosine phenol lyase activity istutA; the at least one gene encoding a polypeptide having chorismatelyase activity is ubiC; the at least one gene encoding a polypeptidehaving p-hydroxybenzoate decarboxylase activity is kpdBCD; the at leastone gene encoding a polypeptide having p-hydroxybenzoate hydroxylaseactivity is pobA; the at least one gene encoding a polypeptide havingprotocatechuate decarboxylase activity is aroY; and/or the at least onegene encoding a polypeptide having 3-dehydroshikimate dehydrataseactivity is aroZ, quiC or qsuB.

In certain embodiments, the recombinant host cell comprises a plasmidcombination selected from the group consisting of: pY3 and pTutA-pPh(CAT2); pSDC-PchB-EntC and pPh (CAT3); pUbiC-Kpd and pPh (CAT4);pUbiC-PobA and pAroY (CAT5); pQsuB-AroY-CatA (MA1); pY3 andpTutA-pPh-CatA (MA2); pSDC-PchB-EntC and pPh-CatA (MA3); pUbiC-Kpd andpPh-CatA (MA4); pUbiC-PobA and pAroY-CatA (MA5); and pUbiC-PobA;pQsuB-AroY-CatA (MAF); pTyrAfbr-TutA (PHI); pSDC-PchB-EntC (PH2);pUbiC-Kpd (PH3); pTyrAfbr-TutA and pSDC-PchB-EntC (PHF1); pTyrAfbr-TutAand pUbiC-Kpd (PHF2); pSDC-PchB-EntC and pUbiC-Kpd (PHF3); andpTyrAfbr-TutA, pSDC-PchB-EntC, and pUbiC-Kpd (PHF4).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAand comprises a plasmid combination selected from the group consistingof: pY3 and pTutA-pPh (CAT2); pSDC-PchB-EntC and pPh (CAT3); pUbiC-Kpdand pPh (CAT4); and pUbiC-PobA and pAroY (CAT5).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAand comprises a plasmid combination selected from the group consistingof: pQsuB-AroY-CatA (MA1); pY3 and pTutA-pPh-CatA (MA2); pSDC-PchB-EntCand pPh-CatA (MA3); pUbiC-Kpd and pPh-CatA (MA4); pUbiC-PobA andpAroY-CatA (MA5); and pUbiC-PobA and pQsuB-AroY-CatA (MAF).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF and comprises a plasmid combination selected from the groupconsisting of: pQsuB-AroY-CatA (MA1); pY3 and pTutA-pPh-CatA (MA2);pSDC-PchB-EntC and pPh-CatA (MA3); pUbiC-Kpd and pPh-CatA (MA4);pUbiC-PobA and pAroY-CatA (MA5); and pUbiC-PobA and pQsuB-AroY-CatA(MAF).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF Δcrr and comprises a plasmid combination selected from thegroup consisting of: pQsuB-AroY-CatA (MA1); pY3 and pTutA-pPh-CatA(MA2); pSDC-PchB-EntC and pPh-CatA (MA3); pUbiC-Kpd and pPh-CatA (MA4);pUbiC-PobA and pAroY-CatA (MA5); and pUbiC-PobA and pQsuB-AroY-CatA(MAF).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF Δcrr and comprises a plasmid combination pQsuB-AroY-CatA(MA1).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF Δcrr and comprises a plasmid combination pUbiC-PobA andpQsuB-AroY-CatA (MAF).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF Δcrr and comprises a plasmid combination selected from thegroup consisting of: pTyrAfbr-TutA (PH1); pSDC-PchB-EntC (PH2);pUbiC-Kpd (PH3); pTyrAfbr-TutA and pSDC-PchB-EntC (PHF1); pTyrAfbr-TutAand pUbiC-Kpd (PHF2); pSDC-PchB-EntC and pUbiC-Kpd (PHF3); andpTyrAfbr-TutA, pSDC-PchB-EntC, and pUbiC-Kpd (PHF4).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAand comprises a plasmid combination selected from the group consistingof: pTyrAfbr-TutA (PH1); pSDC-PchB-EntC (PH2); pUbiC-Kpd (PH3);pTyrAfbr-TutA and pSDC-PchB-EntC (PHF1); pTyrAfbr-TutA and pUbiC-Kpd(PHF2); pSDC-PchB-EntC and pUbiC-Kpd (PHF3); and pTyrAfbr-TutA,pSDC-PchB-EntC, and pUbiC-Kpd (PHF4).

It will be appreciated that the present embodiments are not limited tothe specific genes mentioned above, but will encompass any suitablehomologs of such genes that may be obtained by standard methods. Methodsof obtaining homologs to these genes using sequence-dependent protocolsare well known in the art. Examples of sequence-dependent protocolsinclude, but are not limited to, methods of nucleic acid hybridization,and methods of DNA and RNA amplification as exemplified by various usesof nucleic acid amplification technologies (e.g., polymerase chainreaction (PCR)). For example, genes encoding homologs of thepolypeptides that alone or in combination have the above-mentionedactivities could be isolated directly by using all or a portion of theknown sequences as DNA hybridization probes to screen libraries from anydesired plant, fungi, yeast, or bacteria using methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon theliterature nucleic acid sequences can be designed and synthesized bymethods known in the art. Moreover, the entire sequences can be useddirectly to synthesize DNA probes by methods known to those skilled inthe art, such as random primers DNA labeling, nick translation, orend-labeling techniques or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part of or full length of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length cDNA or genomic fragments underconditions of appropriate stringency.

Synthetic Primers for Cloning

Described herein are methods for the microbial production of biochemicalproducts, such as phenol, catechol, and muconic acid, from renewablesubstrates using recombinant host cells. Specifically, embodiments mayinvolve the cloning and incorporation of genes encoding polypeptideshaving the above described activity(ies) into a single host organism andthe use of those organisms to convert renewable resources such asglucose, for example, to phenol, catechol and muconic acid. As discussedbelow, synthetic DNA primers used to clone the aforementioned genes areknown in the art.

In certain embodiments, the gene encoding a polypeptide havingisochorismate synthase activity is entC. As described in the Examples,the entC gene was cloned using two synthetic nucleotide primerscontaining the following sequences: ATA GGA TCC AGG AGG ATA AAT AAT GGATAC GTC ACT GGC TGA (SEQ ID NO: 12) and ATT CTG CAG TTA ATG CAA TCC AAAAAC GTT (SEQ ID NO: 13). Accordingly, in certain embodiments, at leastone synthetic oligonucleotide primer comprising/consisting of a sequencehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:12and/or SEQ ID NO: 13 is used to clone a gene encoding a polypeptidehaving isochorismate synthase activity (e.g., entC).

In certain embodiments, the gene encoding a polypeptide havingisochorismate pyruvate lyase activity is pchB. As described in theExamples, the pchB gene was cloned using two synthetic nucleotideprimers containing the following sequences: AAT ATC TAG ATT CCC GAG AGGTTG CAT GAT GAA AAC T (SEQ ID NO: 14) and ATT GGA TCC TTA TGC GGC ACCCCG TGT CTG G (SEQ ID NO: 15). Accordingly, in certain embodiments, atleast one synthetic oligonucleotide primer comprising/consisting of asequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQID NO:14 and/or SEQ ID NO: 15 is used to clone a gene encoding apolypeptide having isochorismate pyruvate lyase activity (e.g., pchB).

In certain embodiments, the gene encoding a polypeptide havingsalicylate decarboxylase activity is SDC. As described in the Examples,the SDC gene was cloned using two synthetic nucleotide primerscontaining the following sequences: ATA GAA TTC AGG AGG ATA AAT AAT GCGTGG TAA AGT TAG CCT G (SEQ ID NO: 16) and ATT GGA TCC TTA GGC TTC GCTGTC ATA GAA T (SEQ ID NO: 17). Accordingly, in certain embodiments, atleast one synthetic oligonucleotide primer comprising/consisting of asequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQID NO:16 and/or SEQ ID NO: 17 is to clone a gene encoding a polypeptidehaving salicylate decarboxylase activity (e.g., SDC).

In certain embodiments, the gene encoding a polypeptide having phenolhydroxylase activity is phKLMNOP. As described in the Examples, thephKLMNOP gene was cloned using two synthetic nucleotide primerscontaining the following sequences: ATA TCT AGA AGG AGG ATA AAT AGA GCTCGT GCT GCC TCA CGA (SEQ ID NO: 18) and ATT CCT GCA GGA TGC CCA TGA CTATAT CTT CTT GAA CAG GGC (SEQ ID NO: 19). Accordingly, in certainembodiments, at least one synthetic oligonucleotide primercomprising/consisting of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:18 and/or SEQ ID NO: 19 is used to clonea gene encoding a polypeptide having phenol hydroxylase activity (e.g.,phKLMNOP).

In certain embodiments, the gene encoding a polypeptide having catechol1,2-dioxygenase activity is catA. As described in the Examples, the catAgene was cloned using two synthetic nucleotide primers containing thefollowing sequences: ATA AGA TCT AGG AGG ATA AAT AAT GAC CGT GAA AAT TTCCCA C (SEQ ID NO: 20) and ATT TCT AGA TCA GCC CTC CTG CAA CGC (SEQ IDNO: 21). Accordingly, in certain embodiments, at least one syntheticoligonucleotide primer comprising/consisting of a sequence having atleast about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:20 and/or SEQID NO: 21 is used to clone a gene encoding a polypeptide having catechol1,2-dioxygenase activity (e.g., catA).

In certain embodiments, the gene encoding a polypeptide having tyrosinephenol lyase activity is tutA. As described in the Examples, the tutAgene was cloned using two synthetic nucleotide primers containing thefollowing sequences: ATA GAA TTC AGG AGG ATA AAT AAT GAA TTA TCC GGC AGAACC (SEQ ID NO: 22) and ATT TCT AGA TTA GAT ATA GTC AAA GCG TGC AGT A(SEQ ID NO: 23). Accordingly, in certain embodiments, at least onesynthetic oligonucleotide primer comprising/consisting of a sequencehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:22and/or SEQ ID NO: 23 is used to clone a gene encoding a polypeptidehaving tyrosine phenol lyase activity (e.g., tutA).

In certain embodiments, the gene encoding a polypeptide havingchorismate pyruvate lyase activity is ubiC. As described in theExamples, the ubiC gene was cloned using two synthetic nucleotideprimers containing the following sequences: ATA GAA TTC AGG AGG ATA AATAAT GTC ACA CCC CGC GTT AAC G (SEQ ID NO: 24) and ATT AGA TCT TTA GTACAA CGG TGA CGC CGG TAA A (SEQ ID NO: 25). Accordingly, in certainembodiments, at least one synthetic oligonucleotide primercomprising/consisting of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:24 and/or SEQ ID NO: 25 is used to clonea gene encoding a polypeptide having chorismate synthase activity (e.g.,ubiC).

In certain embodiments, the gene encoding a polypeptide havingp-hydroxybenzoate decarboxylase activity is kpdBCD. As described in theExamples, the kpdBCD gene was cloned using two synthetic nucleotideprimers containing the following sequences: ATA GGA TCC CCC GTC CGG AGAGGG TAA TTT AAA TAT AAA GTT CG (SEQ ID NO: 26) and ATT AAG CTT CTT AGCGGG CCC CTT TAT TAA CGC T (SEQ ID NO: 27). Accordingly, in certainembodiments, at least one synthetic oligonucleotide primercomprising/consisting of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:26 and/or SEQ ID NO: 27 is used to clonea gene encoding a polypeptide having p-hydroxybenzoate decarboxylaseactivity (e.g., kpdBCD).

In certain embodiments, the gene encoding a polypeptide havingp-hydroxybenzoate hydroxylase activity is pobA. As described in theExamples, the pobA gene was cloned using two synthetic nucleotideprimers containing the following sequences: ATA TCT AGA AGG AGG ATA AATAAT GAA GAC TCA AGT CGC CAT CAT CG (SEQ ID NO: 28) and TAT AAG CTT TACTCG ATT TCC TCG TAG GGC (SEQ ID NO: 29). Accordingly, in certainembodiments, at least one synthetic oligonucleotide primercomprising/consisting of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:28 and/or SEQ ID NO: 29 is used to clonea gene encoding a polypeptide having p-hydroxybenzoate hydroxylaseactivity (e.g., pobA).

In certain embodiments, the gene encoding a polypeptide havingprotocatechuate decarboxylase activity is aroY. As described in theExamples, the aroY gene was cloned using two synthetic nucleotideprimers containing the following sequences: ATA AAG CTT AGG AGG ATA AATAAT GAC CGC ACC GAT TC (SEQ ID NO: 30) and ATT CTC GAG TTA TTT TGC GCTACC CTG GTT TTT TTC CAG C (SEQ ID NO: 31 Accordingly, in certainembodiments, at least one synthetic oligonucleotide primercomprising/consisting of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:30 and/or SEQ ID NO: 31 is used to clonea gene encoding a polypeptide having protocatechuate decarboxylaseactivity (e.g., aroY).

In certain embodiments, the gene encoding a polypeptide having3-dehydroshikimate dehydratase activity is qsuB. As described in theExamples, the qsuB gene was cloned using two synthetic nucleotideprimers containing the following sequences: ATA GGA TCC AGG AGG ATA AATAAT GCG TAC ATC CAT TGC CAC TGT TTG (SEQ ID NO: 32) and ATT AAG CTT CTAGTT TGG GAT TCC CCG CTC GA (SEQ ID NO: 33). Accordingly, in certainembodiments, at least one synthetic oligonucleotide primercomprising/consisting of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:32 and/or SEQ ID NO: 33 is used to clonea gene encoding a polypeptide having 3-dehydroshikimate dehydrataseactivity (e.g., qsuB).

As described herein, multiple genes may be cloned and expressed in asingle host organism for use in the methods described herein. Thesegenes may be expressed using separate vectors or the same vector.Described below are primers used to generate a gene series forexpression in a single vector. Also discussed below are varioussynthetic plasmid backbones, which comprise multiple elements andprimers used to clone such series are described.

In certain embodiments, a gene series encoding polypeptides havingchorismate synthase, 3-phosphoshikimate 1-carboxyvinyltransferase andshikimate kinase activities is composed of aroC-aroA-aroL. As describedin the Examples, the aroC-aroA-aroL gene series was cloned using GibsonAssembly with two synthetic nucleotide primers containing the followingsequences: AGA TCT AAA GGA GGC CAT CCA TGG CTG GAA ACA CAA TTG G (SEQ IDNO: 34) and ATG CCT GGA GAT CCT TAC TCG AGT TTG GAT CCT C (SEQ ID NO:35). Accordingly, in certain embodiments, at least one syntheticoligonucleotide primer comprising/consisting of a sequence having atleast about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:34 and/or SEQID NO: 35 is used to clone a gene encoding a series of polypeptideshaving 3-phosphoshikimate 1-carboxyvinyltransferase, and shikimatekinase activities.

In certain embodiments, DNA encoding a synthetic plasmid backbone iscomposed of p15A ori, lacI repressor, lacUV5 promoter, and Ampicillinresistance marker. As described in the Examples, said series was clonedusing Gibson Assembly with two synthetic nucleotide primers containingthe following sequences: GAG GAT CCA AAC TCG AGT AAG GAT CTC CAG GCA T(SEQ ID NO: 36) and CCA ATT GTG TTT CCA GCC ATG GAT GGC CTC CTT TAG ATCT (SEQ ID NO: 37). Accordingly, in certain embodiments, at least onesynthetic oligonucleotide primer comprising/consisting of a sequencehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:36and/or SEQ ID NO: 37 is used to clone the above described plasmidbackbone.

In certain embodiments, DNA encoding a synthetic plasmid backbone iscomposed of p15A ori, lacI repressor, lacUV5 promoter, and Ampicillinresistance marker. As described in the Examples, said series was clonedusing Gibson Assembly with two synthetic nucleotide primers containingthe following sequences: CTG CAC GCT TTG ACT ATA TCT AAG GAT CCA AAC TCGAGT AAG G (SEQ ID NO: 38) and CAT GGA TGG CCT CCT AGA TCT TTT GAA TTCTGA AAT TGT TAT C (SEQ ID NO: 39). Accordingly, in certain embodiments,at least one synthetic oligonucleotide primer comprising/consisting of asequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQID NO:38 and/or SEQ ID NO: 39 is used to clone a the above describedplasmid backbone.

In certain embodiments, the gene encoding feedback resistant chorismatemutase/prephenate dehydrogenase activity is composed of tyrA^(fbr). Asdescribed in the Examples, tyrA^(fbr) was cloned using Gibson Assemblywith two synthetic nucleotide primers containing the followingsequences: GAT AAC AAT TTC AGA ATT CAA AAG ATC TAG GAG GCC ATC CAT G(SEQ ID NO: 40) and CGG ATA ATT CAT TAT TTA TCC TCC TTT AGA TCC TTA CTGGCG ATT (SEQ ID NO: 41). Accordingly, in certain embodiments, at leastone synthetic oligonucleotide primer comprising/consisting of a sequencehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:40and/or SEQ ID NO: 41 is used to clone a polypeptide displayingchorismate mutase/prephenate dehydrogenase activity.

In certain embodiments, the gene encoding tyrosine phenol lyase activityis composed of tutA. As described in the Examples, tutA was cloned usingGibson Assembly with two synthetic nucleotide primers containing thefollowing sequences: AAT CGC CAG TAA GGA TCT AAA GGA GGA TAA ATA ATG AATTAT CCG (SEQ ID NO: 42) and CCT TAC TCG AGT TTG GAT CCT TAG ATA TAG TCAAAG CGT GCA G (SEQ ID NO: 43). Accordingly, in certain embodiments, atleast one synthetic oligonucleotide primer comprising/consisting of asequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQID NO:42 and/or SEQ ID NO: 43 is used to clone a polypeptide displayingtyrosine phenol lyase activity.

In certain embodiments, the gene series encoding polypeptides havingsalicylate decarboxylase, isochorismate pyruvate lyase, andisochorismate synthase activities is composed of SDC-pchB-entC. Asdescribed in the Examples, the SDC-pchB-entC gene series was clonedusing Gibson Assembly with two synthetic nucleotide primers containingthe following sequences: AAG GAG GCC ATC CAT GCG TGG TAA AGT TAG C (SEQID NO: 44) and GTT TGG ATC CTT AAT GCA ATC CAA AAA CG (SEQ ID NO: 45).Accordingly, in certain embodiments, at least one syntheticoligonucleotide primer comprising/consisting of a sequence having atleast about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:44 and/or SEQID NO: 45 is used to clone a gene series encoding polypeptides havingsalicylate decarboxylase, isochorismate pyruvate lyase, andisochorismate synthase activities.

In certain embodiments, DNA encoding a synthetic plasmid backbone iscomposed of pBBR1 ori, lacI repressor, lacUV5 promoter, andChloramphenicol resistance marker. As described in the Examples, saidseries was cloned using Gibson Assembly with two synthetic nucleotideprimers containing the following sequences: ATT GCA TTA AGG ATC CAA ACTCGA GTA AG (SEQ ID NO: 46) and CTT TAC CAC GCA TGG ATG GCC TCC TTT AGATC (SEQ ID NO: 47). Accordingly, in certain embodiments, at least onesynthetic oligonucleotide primer comprising/consisting of a sequencehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:46and/or SEQ ID NO: 47 is used to clone the above described plasmidbackbone.

In certain embodiments, DNA encoding a polypeptide having kanamycinresistance in place of chorismate mutase/prephenate dehydrogenaseactivity is composed of ΔpheA::FRT-Kan^(R)-FRT. As described in theExamples, the ΔpheA::FRT-Kan^(R)-FRT DNA cassette was cloned using twosynthetic nucleotide primers containing the following sequences: CGT GTGAAA CAG AAT GCG AAG ACG AAC AAT A (SEQ ID NO: 48) and TAA TCC AGT GCCGGA TGA TTC ACA TCA TC (SEQ ID NO: 49). Accordingly, in certainembodiments, at least one synthetic oligonucleotide primercomprising/consisting of a sequence having at least about 50%, 55%, 60%,65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO:48 and/or SEQ ID NO: 49 is used to clonea DNA cassette encoding ΔpheA::FRT-Kan^(R)-FRT.

In certain embodiments, DNA encoding a polypeptide having kanamycinresistance in place of pyruvate kinase II activity is composed ofΔpykA::FRT-Kan^(R)-FRT. As described in the Examples, theΔpykA::FRT-Kan^(R)-FRT DNA cassette was cloned using two syntheticnucleotide primers containing the following sequences: ATC GCG GCG TTATTT CAT TCG GAT T (SEQ ID NO: 50) and AAC TGT AGG CCG GAT GTG GC (SEQ IDNO: 51). Accordingly, in certain embodiments, at least one syntheticoligonucleotide primer comprising/consisting of a sequence having atleast about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:50 and/or SEQID NO: 51 is used to clone a DNA cassette encodingΔpykA::FRT-Kan^(R)-FRT.

In certain embodiments, DNA encoding a polypeptide having kanamycinresistance in place of pyruvate kinase activity is composed ofΔpykF::FRT-Kan^(R)-FRT. As described in the Examples, theΔpykF::FRT-Kan^(R)-FRT DNA cassette was cloned using two syntheticnucleotide primers containing the following sequences: GCG AGG CAC CACCAC TTT CG (SEQ ID NO: 52) and AGC GCC CAT CAG GGC G (SEQ ID NO: 53).Accordingly, in certain embodiments, at least one syntheticoligonucleotide primer comprising/consisting of a sequence having atleast about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:52 and/or SEQID NO: 53 is used to clone a DNA cassette encodingΔpykF::FRT-Kan^(R)-FRT

In certain embodiments, the DNA encoding a polypeptide having kanamycinresistance in place of IIA^(glc) activity is composed ofΔcrr::FRT-Kan^(R)-FRT. As described in the Examples, theΔcrr::FRT-Kan^(R)-FRT DNA cassette was cloned using two syntheticnucleotide primers containing the following sequences: CTA TGA GCG CCATTT CTA TCC CGC GC (SEQ ID NO: 54) and CCT GAA AGG GAC TGG CGA CCT G(SEQ ID NO: 55). Accordingly, in certain embodiments, at least onesynthetic oligonucleotide primer comprising/consisting of a sequencehaving at least about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:54and/or SEQ ID NO: 55 is used to clone a DNA cassette encodingΔcrr::FRT-Kan^(R)-FRT.

Exemplary Recombinant Host Cells, Compositions and Kits According to Oneor More Embodiments

Certain embodiments provide an expression cassette described herein. Incertain embodiments, the expression cassette further comprises one ormore promoters. In certain embodiments, the expression cassette furthercomprises one or more regulatory elements.

Certain embodiments provide a vector described herein (e.g., a plasmiddescribed herein, such as in the Examples, Figures or Tables).

Certain embodiments provide a synthetic oligonucleotide primer describedherein.

Certain embodiments provide a recombinant host cell as described herein(e.g., as described in the Examples, Figures or Tables).

In certain embodiments, the recombinant host cell is Escherichia coli.In certain embodiments, the recombinant host cell is E. coli NST74, E.coli NST74 ΔpheA, E. coli NST74 ΔpheA ΔpykA ΔpykF, or E. coli NST74ΔpheA ΔpykA ΔpykF Δcrr.

In certain embodiments, the recombinant host cell comprises a plasmidcombination selected from the group consisting of: pY3 and pTutA-pPh(CAT2); pSDC-PchB-EntC and pPh (CAT3); pUbiC-Kpd and pPh (CAT4);pUbiC-PobA and pAroY (CAT5); pQsuB-AroY-CatA (MA1); pY3 andpTutA-pPh-CatA (MA2); pSDC-PchB-EntC and pPh-CatA (MA3); pUbiC-Kpd andpPh-CatA (MA4); pUbiC-PobA and pAroY-CatA (MA5); and pUbiC-PobA;pQsuB-AroY-CatA (MAF); pTyrAfbr-TutA (PHI); pSDC-PchB-EntC (PH2);pUbiC-Kpd (PH3); pTyrAfbr-TutA and pSDC-PchB-EntC (PHF1); pTyrAfbr-TutAand pUbiC-Kpd (PHF2); pSDC-PchB-EntC and pUbiC-Kpd (PHF3); andpTyrAfbr-TutA, pSDC-PchB-EntC, and pUbiC-Kpd (PHF4).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAand comprises a plasmid combination selected from the group consistingof: pY3 and pTutA-pPh (CAT2); pSDC-PchB-EntC and pPh (CAT3); pUbiC-Kpdand pPh (CAT4); and pUbiC-PobA and pAroY (CAT5).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAand comprises a plasmid combination selected from the group consistingof: pQsuB-AroY-CatA (MA1); pY3 and pTutA-pPh-CatA (MA2); pSDC-PchB-EntCand pPh-CatA (MA3); pUbiC-Kpd and pPh-CatA (MA4); pUbiC-PobA andpAroY-CatA (MA5); and pUbiC-PobA and pQsuB-AroY-CatA (MAF).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF and comprises a plasmid combination selected from the groupconsisting of: pQsuB-AroY-CatA (MA1); pY3 and pTutA-pPh-CatA (MA2);pSDC-PchB-EntC and pPh-CatA (MA3); pUbiC-Kpd and pPh-CatA (MA4);pUbiC-PobA and pAroY-CatA (MA5); and pUbiC-PobA and pQsuB-AroY-CatA(MAF).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF Δcrr and comprises a plasmid combination selected from thegroup consisting of: pQsuB-AroY-CatA (MA1); pY3 and pTutA-pPh-CatA(MA2); pSDC-PchB-EntC and pPh-CatA (MA3); pUbiC-Kpd and pPh-CatA (MA4);pUbiC-PobA and pAroY-CatA (MA5); and pUbiC-PobA and pQsuB-AroY-CatA(MAF).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF Δcrr and comprises a plasmid combination pQsuB-AroY-CatA(MA1).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF Δcrr and comprises a plasmid combination pUbiC-PobA andpQsuB-AroY-CatA (MAF).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAΔpykA ΔpykF Δcrr and comprises a plasmid combination selected from thegroup consisting of: pTyrAfbr-TutA (PH1); pSDC-PchB-EntC (PH2);pUbiC-Kpd (PH3); pTyrAfbr-TutA and pSDC-PchB-EntC (PHF1); pTyrAfbr-TutAand pUbiC-Kpd (PHF2); pSDC-PchB-EntC and pUbiC-Kpd (PHF3); andpTyrAfbr-TutA, pSDC-PchB-EntC, and pUbiC-Kpd (PHF4).

In certain embodiments, the recombinant host cell is E. coli NST74 ΔpheAand comprises a plasmid combination selected from the group consistingof: pTyrAfbr-TutA (PH1); pSDC-PchB-EntC (PH2); pUbiC-Kpd (PH3);pTyrAfbr-TutA and pSDC-PchB-EntC (PHF1); pTyrAfbr-TutA and pUbiC-Kpd(PHF2); pSDC-PchB-EntC and pUbiC-Kpd (PHF3); and pTyrAfbr-TutA,pSDC-PchB-EntC, and pUbiC-Kpd (PHF4).

Certain embodiments provide a composition comprising a recombinant hostcell described herein and a carrier. In certain embodiments, thecomposition further comprises a growth supplement (growth media orbroth).

Further provided are kits for practicing the present methods. Forexample, certain embodiments provide a kit comprising a recombinant hostcell described herein and instructions for generating a biochemicalproduct using the recombinant cell (e.g., instructions to practice amethod described herein).

Certain Definitions

The term “isochorismate synthase activity” refers to the ability of aprotein to catalyze the direct conversion of chorismate toisochorismate.

The term “isochorismate pyruvate lyase activity” refers to the abilityof a protein to catalyze the direct conversion of isochorismate tosalicylate.

The term “salicylate decarboxylase activity” refers to the ability of aprotein to catalyze the direct conversion of salicylate to phenol.

The term “phenol 2-monooxygenase activity” refers to the ability of aprotein to catalyze the direct conversion of phenol to catechol.

The term “catechol-1,2-dioxygenase activity” refers to the ability of aprotein to catalyze the direct conversion of catechol to cis,cis-muconicacid.

The term “tyrosine phenol lyase activity” refers to the ability of aprotein to catalyze the direct conversion of tyrosine to phenol.

The term “chorismate lyase activity” or “chorismate pyruvate lyaseactivity” refers to the ability of a protein to catalyze the directconversion of chorismate to p-hydroxybenzoate.

The term “p-hydroxybenzoate decarboxylase activity” refers to theability of a protein to catalyze the direct conversion ofp-hydroxybenzoate to phenol.

The term “p-hydroxybenzoate hydroxylase activity” refers to the abilityof a protein to catalyze the direct conversion of p-hydroxybenzoate toprotocatechuate.

The term “protocatechuate decarboxylase activity” refers to the abilityof a protein to catalyze the direct conversion of protocatechuate tocatechol.

The term “3-dehydroshikimate dehydratase activity” refers to the abilityof a protein to catalyze the direct conversion of 3-dehydroshikimate toprotocatechuate.

The term “chorismate mutase activity” refers to the ability of a proteinto catalyze the direct conversion of chorismate to prephenate.

The term “prephenate dehydrogenase activity” refers to the ability of aprotein to catalyze the direct conversion of prephenate to4-hydroxy-phenylpyruvate.

The term “3-DHS” refers to 3-dehydroshikimate.

The term “PCA” refers to protocatechuate.

The term “Phe” refers to L-phenylalanine.

The term “Tyr” refers to L-tyrosine.

The term “Trp” refers to L-tryptophan.

The term “pHBA” refers to p-hydroxybenzoate.

The term “PCA” refers to protocatechuate.

The term “MA” refers to muconic acid.

The term “1,2,3-THB” refers to 1,2,3-trihydroxybenzene.

The term “CDO” refers to catechol-1,2-dioxygenase.

The term “PH” refers to phenol hydroxylase.

The term “TPL” refers to tyrosine phenol lyase.

The term “ToMo” refers to toluene/o-xylene monooxygenase.

The term “host” or “recombinant host” refers any organism (e.g.,microorganism or plant) or suitable cell line, such as a strain ofbacteria, for example, into which genes can be transferred to impartdesired genetic attributes and functions.

The term “recombinant pathway” refers to a pathway that has beenmodified using recombinant techniques (e.g., the pathway comprises arecombinant protein that is not endogenously expressed by the host).

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al. (1991) Nucl. Acids Res., 19:508;Ohtsuka et al. (1985) JBC, 260:2605; Rossolini et al. (1994) Mol. Cell.Probes, 8:91. A “nucleic acid fragment” is a fraction of a given nucleicacid molecule. Deoxyribonucleic acid (DNA) in the majority of organismsis the genetic material while ribonucleic acid (RNA) is involved in thetransfer of information contained within DNA into proteins. The term“nucleotide sequence” refers to a polymer of DNA or RNA that can besingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases capable of incorporation into DNA or RNApolymers. The terms “nucleic acid,” “nucleic acid molecule,” “nucleicacid fragment,” “nucleic acid sequence or segment,” or “polynucleotide”may also be used interchangeably with gene, cDNA, DNA and RNA encoded bya gene.

By “portion” or “fragment,” as it relates to a nucleic acid molecule,sequence or segment, when it is linked to other sequences forexpression, is meant a sequence having, e.g., at least about 80nucleotides, at least about 150 nucleotides, or at least about 400nucleotides. If not employed for expressing, a “portion” or “fragment”means, e.g., at least about 9, at least about 12, at least about 15, orat least about 20, consecutive nucleotides, e.g., probes and primers(oligonucleotides), corresponding to the nucleotide sequence of thenucleic acid molecules described herein.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

Isolated or substantially purified nucleic acid or protein compositionsare also described herein. An “isolated” or “purified” DNA molecule oran “isolated” or “purified” polypeptide is a DNA molecule or polypeptidethat exists apart from its native environment and is therefore not aproduct of nature. An isolated DNA molecule or polypeptide may exist ina purified form or may exist in a non-native environment such as, forexample, a transgenic host cell. For example, an “isolated” or“purified” nucleic acid molecule or protein, or biologically activeportion thereof, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. In one embodiment, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. For example, in variousembodiments, the isolated nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. A protein that issubstantially free of cellular material includes preparations of proteinor polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight)of contaminating protein. When a protein, or biologically active portionthereof, is recombinantly produced, culture medium may represent, e.g.,less than about 30%, 20%, 10%, or 5% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals. Fragments and variantsof the disclosed nucleotide sequences and proteins or partial-lengthproteins encoded thereby are also described herein. By “fragment” or“portion” is meant a full length or less than full length of thenucleotide sequence encoding, or the amino acid sequence of, apolypeptide or protein.

“Naturally occurring” or “wildtype” is used to describe an object thatcan be found in nature as distinct from being artificially produced. Forexample, a protein or nucleotide sequence present in an organism(including a virus), which can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory, isnaturally occurring.

A “variant” of a molecule is a sequence that is substantially similar tothe sequence of the native molecule. For nucleotide sequences, variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis that encode the native protein, as wellas those that encode a polypeptide having amino acid substitutions.Generally, nucleotide sequence variants will have, e.g., at least about40, 50 or 60 to 70%, or e.g., about 71%, 72%, 73%, 74%, 75%, 76%, 77%,or 78% to 79% or generally at least 80%, e.g., 81%-84%, at least 85%,e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to98%, sequence identity to the native (endogenous) nucleotide sequence.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance, the codons CGT, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except ATG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

“Recombinant DNA molecule” is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook andRussell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (3^(rd) edition, 2001).

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Genes include coding sequencesand/or the regulatory sequences required for their expression. Forexample, gene refers to a nucleic acid fragment that expresses mRNA,functional RNA, or a specific protein, including its regulatorysequences. Genes also include nonexpressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters. Inaddition, a “gene” or a “recombinant gene” refers to a nucleic acidmolecule comprising an open reading frame and including at least oneexon and (optionally) an intron sequence. The term “intron” refers to aDNA sequence present in a given gene which is not translated intoprotein and is generally found between exons. “Native gene” or “wildtype gene” refers to a gene as found in nature with its own regulatorysequences. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. “Foreign gene or heterologousgene” refers to a gene not normally found in the host organism but thatis introduced into the host organism by gene transfer. Foreign genes cancomprise native genes inserted into a non-native organism, or chimericgenes.

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous nucleic acid,” each refer to a sequence that originatesfrom a source foreign to the particular host cell or, if from the samesource, is modified from its original form. Thus, a heterologous gene ina host cell includes a gene that is endogenous to the particular hostcell but has been modified.

The terms also include non-naturally occurring multiple copies of anaturally occurring DNA sequence. Thus, the terms refer to a DNA segmentthat is foreign or heterologous to the cell, or homologous to the cellbut in a position within the host cell nucleic acid in which the elementis not ordinarily found. Exogenous DNA segments are expressed to yieldexogenous polypeptides.

A “homologous” DNA sequence is a DNA sequence that is naturallyassociated with a host cell into which it is introduced.

“Genome” refers to the complete genetic material of an organism.

A “vector” is defined to include, inter alia, any viral vector, plasmid,cosmid, phage or binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.,autonomous replicating plasmid with an origin of replication).

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance, hygromycin resistance or ampicillin resistance.

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA, in the senseor antisense direction. The expression cassette comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression cassette may also be one that isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. The expression of the nucleotide sequencein the expression cassette may be under the control of a constitutivepromoter or of an inducible promoter that initiates transcription onlywhen the host cell is exposed to some particular external stimulus. Inthe case of a multicellular organism, the promoter can also be specificto a particular tissue or organ or stage of development.

Such expression cassettes will comprise the transcriptional initiationregion linked to a nucleotide sequence of interest. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the gene of interest to be under the transcriptional regulation ofthe regulatory regions. The expression cassette may additionally containselectable marker genes.

The term “RNA transcript” refers to the product resulting from RNApolymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

“Regulatory sequences” and “suitable regulatory sequences” each refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences includeenhancers, promoters, translation leader sequences, introns, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences that may be a combination of syntheticand natural sequences. As is noted above, the term “suitable regulatorysequences” is not limited to promoters. However, some suitableregulatory sequences may include, but are not limited to constitutivepromoters, tissue-specific promoters, development-specific promoters,inducible promoters and viral promoters.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner et al. (1995) Mol. Biotech. 3:225).

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or even becomprised of synthetic DNA segments. A promoter may also contain DNAsequences that are involved in the binding of protein factors thatcontrol the effectiveness of transcription initiation in response tophysiological or developmental conditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one is affected bythe other. For example, a regulatory DNA sequence is said to be“operably linked to” or “associated with” a DNA sequence that codes foran RNA or a polypeptide if the two sequences are situated such that theregulatory DNA sequence affects expression of the coding DNA sequence(i.e., that the coding sequence or functional RNA is under thetranscriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation in a cell ofan endogenous gene, transgene, as well as the transcription and stableaccumulation of sense (mRNA) or functional RNA. In the case of antisenseconstructs, expression may refer to the transcription of the antisenseDNA only. Expression may also refer to the production of protein.“Overexpession” refers to the production of a gene product in atransgenic organism that exceeds levels of production in the wild-typehost or native organisms.

“Transcription stop fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples oftranscription stop fragments are known to the art.

“Translation stop fragment” refers to nucleotide sequences that containone or more regulatory signals, such as one or more termination codonsin all three frames, capable of terminating translation. Insertion of atranslation stop fragment adjacent to or near the initiation codon atthe 5′ end of the coding sequence will result in no translation orimproper translation. Excision of the translation stop fragment bysite-specific recombination will leave a site-specific sequence in thecoding sequence that does not interfere with proper translation usingthe initiation codon.

The terms “cis-acting sequence” and “cis-acting element” refer to DNA orRNA sequences whose functions require them to be on the same molecule.

The terms “trans-acting sequence” and “trans-acting element” refer toDNA or RNA sequences whose function does not require them to be on thesame molecule.

The following terms are used to describe the sequence relationshipsbetween two or more sequences (e.g., nucleic acids, polynucleotides orpolypeptides): (a) “reference sequence,” (b) “comparison window,” (c)“sequence identity,” (d) “percentage of sequence identity,” and (e)“substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull length cDNA, gene sequence or peptide sequence, or the completecDNA, gene sequence or peptide sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a sequence, wherein the sequence in thecomparison window may comprise additions or deletions (i.e., gaps)compared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. Generally, thecomparison window is at least 20 contiguous nucleotides in length, andoptionally can be 30, 40, 50, 100, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS, 4:11; the local homology algorithm ofSmith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch, (1970) JMB, 48:443; thesearch-for-similarity-method of Pearson and Lipman, (1988) Proc. Natl.Acad. Sci. USA, 85:2444; the algorithm of Karlin and Altschul, (1990)Proc. Natl. Acad. Sci. USA, 87:2264, modified as in Karlin and Altschul,(1993) Proc. Natl. Acad. Sci. USA, 90:5873.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237; Higgins et al. (1989) CABIOS 5:151; Corpet et al.(1988) Nucl. Acids Res. 16:10881; Huang et al. (1992) CABIOS 8:155; andPearson et al. (1994) Meth. Mol. Biol. 24:307. The ALIGN program isbased on the algorithm of Myers and Miller, supra. The BLAST programs ofAltschul et al. (1990) JMB, 215:403; Nucl. Acids Res., 25:3389 (1990),are based on the algorithm of Karlin and Altschul supra.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (available on the worldwide web at ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when the cumulative alignmentscore falls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, less than about0.01, or less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al., supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of100, M=5, N=−4, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as defaults a wordlength (W) of 3, anexpectation (E) of 10, and the BLOSUM62 scoring matrix. See the worldwide web at ncbi.nlm.nih.gov. Alignment may also be performed manuallyby visual inspection.

Comparison of sequences for determination of percent sequence identityto another sequence may be made using the BlastN program (version 1.4.7or later) with its default parameters or any equivalent program. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by the program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to a specifiedpercentage of residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window,as measured by sequence comparison algorithms or by visual inspection.When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typically,this involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percentage of sequenceidentity.

(e)(i) The term “substantial identity” of sequences means that apolynucleotide comprises a sequence that has at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, and atleast 95%, 96%, 97%, 98%, or 99% sequence identity, compared to areference sequence using one of the alignment programs described usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 70%, at least 80%,90%, at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%,97%, 98% or 99%, sequence identity to the reference sequence over aspecified comparison window. Optimal alignment is conducted using thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443 (1970). An indication that two peptide sequences aresubstantially identical is that one peptide is immunologically reactivewith antibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. The thermal melting point(T_(m)) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267; T_(m) 81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. T_(m) is reduced by about 1° C. foreach 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the T_(m) for the specificsequence and its complement at a defined ionic strength and pH. However,severely stringent conditions can utilize a hybridization and/or wash at1, 2, 3, or 4° C. lower than the T_(m); moderately stringent conditionscan utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lowerthan the T_(m); low stringency conditions can utilize a hybridizationand/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the T_(m). Usingthe equation, hybridization and wash compositions, and desiredtemperature, those of ordinary skill will understand that variations inthe stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a temperatureof less than 45° C. (aqueous solution) or 32° C. (formamide solution),the SSC concentration may be increased so that a higher temperature canbe used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology Hybridization with Nucleic Acid Probes, part I chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays” Elsevier, New York (1993). Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the T_(m) for the specific sequence at a defined ionicstrength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.5M (e.g, about 0.01 to 1.0 M), Na ion concentration (or other salts) atpH 7.0 to 8.3, and the temperature is typically at least about 30° C.and at least about 60° C. for long probes (e.g., >50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g., when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

By “variant” polypeptide is intended a polypeptide derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultform, for example, genetic polymorphism or from human manipulation.Methods for such manipulations are generally known in the art.

Thus, the polypeptides described herein may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the polypeptides canbe prepared by mutations in the DNA. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488; Kunkel et al.(1987) Meth. Enzymol. 154:367; U.S. Pat. No. 4,873,192; Walker andGaastra (1983) Techniques in Mol. Biol. (MacMillan Publishing Co., andthe references cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al., Atlas of ProteinSequence and Structure (Natl. Biomed. Res. Found. 1978). Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be used.

Thus, the genes and nucleotide sequences include both the naturallyoccurring sequences as well as mutant forms. Likewise, polypeptidesencompass naturally occurring proteins as well as variations andmodified forms thereof. Such variants will continue to possess thedesired activity. In certain embodiments, the deletions, insertions, andsubstitutions of the polypeptide sequence encompassed herein may notproduce radical changes in the characteristics of the polypeptide.However, when it is difficult to predict the exact effect of thesubstitution, deletion, or insertion in advance of doing so, one skilledin the art will appreciate that the effect will be evaluated by routinescreening assays.

Individual substitutions deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine(R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid(E), Asparagine (N), Glutamine (Q). In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence are also “conservatively modified variations.”

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic”, “recombinant” or“transformed” cells, and organisms comprising transgenic cells arereferred to as “transgenic”, “recombinant” or “transformed” organisms.

“Transformed,” “transgenic,” “transduced” and “recombinant” refer to ahost cell or organism into which a heterologous nucleic acid moleculehas been introduced. The nucleic acid molecule can be stably integratedinto the genome generally known in the art and are disclosed in Sambrookand Russell, supra. See also Innis et al., PCR Protocols, Academic Press(1995); and Gelfand, PCR Strategies, Academic Press (1995); and Innisand Gelfand, PCR Methods Manual, Academic Press (1999). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially mismatchedprimers, and the like. For example, “transformed,” “transformant,” and“transgenic” cells have been through the transformation process andcontain a foreign gene integrated into their chromosome. The term“untransformed” refers to normal cells that have not been through thetransformation process.

The term “endogenous metabolite” refers to a native metabolite naturallypresent in a cell/organism.

Certain embodiments will now be illustrated by the followingnon-limiting Examples.

Example 1. Engineering Novel Pathways and a Synthetic Metabolic Funnelto Enhance Muconic Acid Biosynthesis

Multiple alternative MA biosynthesis pathways have to date beenengineered, each involving different enzyme chemistries and, mostnotably, stemming from precursors further downstream in the shikimicacid pathway, thereby preserving chorismate biosynthesis. To date, nofewer than four alternatives pathways have so far been proposed and/ordeveloped for MA biosynthesis from glucose (Averesch, et al., 2014.Metabolic Engineering Communications. 1, 19-28). For example, Sun et al.recently reported a novel MA biosynthesis pathway utilizing anthranilate(intermediate to Trp) as its immediate endogenous precursor (Sun, etal., 2013b. Applied and environmental microbiology. 79, 4024-30). Bysaid pathway, MA titers of 0.39 g/L were achieved using a mixedglucose/glycerol minimal media (supplemented with Trp to account fordeletion of trpD) in shake flask cultures. The same group also laterengineered another alternative MA pathway, in this case utilizingendogenous 2,3-DHB as its immediate endogenous precursor (Sun, et al.,2014. ChemSusChem. 7, 2478-81). In this case, MA titers of 0.48 g/L werereported using a glucose/glycerol media with yeast extract in shakeflasks cultures. Most recently, the same group has engineered a third,alternative MA pathway that instead stems directly from chorismate andproceeds via the key intermediates isochorismate and salicylate (Lin, etal., 2014. Metabolic engineering. 23). Said pathway resulted in MAtiters reaching 1.45 g/L in shake flask cultures using the sameglucose/glycerol media with yeast extract (in this case to account forPhe and Tyr auxotrophies caused by deletion of pheA and tyrA,respectively, to increase chorismate availability).

In this example, the development of a series of additional, alternativestrategies is reported for MA biosynthesis by: i) constructing a seriesof modular, phenol-derived catechol and MA pathways by linking threerecently-engineered phenol biosynthesis pathways (Thompson, et al.,2016. Biotechnol Bioeng. 113(8), 1745-54) with its subsequent, partialaerobic degradation; ii) engineering a four-step MA pathway fromendogenous chorismate via the intermediates pHBA, PCA, and catechol;and, iii) exploring a synthetic ‘metabolic funnel’ as a novelengineering strategy capable of enhancing MA production via the parallelco-expression of two distinct yet converging MA pathways. All five ofthe proposed strategies importantly circumvent the auxotrophiclimitations experienced via the original, ‘3DHS-derived’ route, while byusing a synthetic ‘metabolic funnel’ it is demonstrated how MA titersand yields can be improved relative to single pathway controls.

Materials and Methods Strains and Media

All strains used in this study are listed in Table 1. E. coli NEB10-beta(New England Biolabs (NEB); Ipswich, Mass.) was used for all cloning andplasmid maintenance. E. coli NST74 (ATCC 31884) was obtained from theAmerican Type Culture Collection (ATCC; Manassas, Va.) and served as theparent strain in this study. E. coli JW2580-1, JW1843-2, JW1666-3, andJW2410-1 were obtained from the Coli Genetic Stock Center (CGSC; NewHaven, Conn.) and served as the genetic source for the pheA::Kan^(R),pykA::Kan^(R), pykF::Kan^(R), and crr::Kan^(R) cassettes, respectively.E. coli BW25113 was obtained from the CGSC and served as the geneticsource for ubiC and entC. Citrobacter braakii (ATCC 29063) was obtainedfrom the ATCC and served as the genetic source for tutA. Klebsiellapneumoniae PZH572 (ATCC 25955) was obtained from the ATCC and served asthe genetic source of kpdBCD and aroY. Pseudomonas aeruginosa PAO1 (DSMZ22644) was obtained from the Leibniz Institute German Collection ofMicroorganisms and Cell Cultures and served as the genetic source ofpobA and pchB. P. stutzeri OX1 (ATCC BAA-172) was obtained from the ATCCand served as the genetic source of phKLMNOP Pseudomonas putida KT2440(ATCC 47054) was obtained from the ATCC and served as the genetic sourcefor catA. Corynebacterium glutamicum (ATCC 13032) was obtained from theATCC and served as the genetic source of qsuB.

Seed cultures of E. coli strains were cultured in Luria-Bertani (LB)broth at 32° C. and supplemented with 100 mg/L ampicillin and/or 35 mg/Lkanamycin, as appropriate. For catechol and MA biosynthesis, shakeflasks were cultured at 32° C. in M9M minimal media supplemented withappropriate antibiotics. M9M was composed of the following (in g/L):Na₂HPO₄ (6), KH₂PO₄ (3), NaCl (0.5), NH₄Cl (2), MgSO₄.7H₂O (0.493),CaCl₂.2H₂O (0.0147), and glucose (20). Trace elements were supplementedas follows (in mg/L): (NH₄)₆Mo₇O₂₄.4H₂O (0.37), H₃BO₃(2.5), CoCl₂.6H₂O(0.714), CuSO₄ (0.16), MnCl₂.4H₂O (1.6), ZnSO₄.7H₂O (0.288), FeCl₃(0.05).

Plasmid Construction

All plasmids used and developed in this study are listed in Table 1. Allgenes were PCR amplified with Q5 High-Fidelity DNA Polymerase (NEB) anda BioRad iCycler, according to manufacturer protocols. Custom DNAoligonucleotide primers were synthesized by Integrated DNA Technologies(IDT, Coralville Iowa) and are listed in Table 2. Genomic DNA (gDNA)templates were prepared using the ZR Fungal/Bacterial DNA MiniPrep kitwhile plasmid DNA was purified using the Zymo Plasmid MiniPrep kit (bothZymo Research, Irvine Calif.). Amplified linear DNA fragments werepurified using the Zymo DNA Clean & Concentrator MiniPrep kit (ZymoResearch). Purified linear DNA and plasmid DNA were digested usingappropriate restriction endonucleases (NEB) and subsequently gelpurified using the Zymoclean Gel DNA Recovery MiniPrep kit (ZymoResearch). Purified digested DNA fragments were ligated using T4 DNALigase (NEB), per manufacturer protocols. Ligation reactions weretransformed into chemically competent E. coli NEB10-beta before platingon LB solid agar media supplemented with appropriate antibiotics forselection. Transformant pools were screened using colony PCR,restriction digest mapping, and finally confirmed by DNA sequencing.

Strain Construction

Chromosomal in-frame deletions of pheA, pykA, pykF, and crr in E. coliNST74 were individually performed using a modified version of theDatsenko and Wanner method (Datsenko, K. A., Wanner, B. L., 2000. etal., Proc Natl Acad Sci USA. 97, 6640-5), as previously described (Pugh,et al., 2014. Process Biochemistry. 49, 1843-1850). ThepheA::FRT-kan^(R)-FRT, pykA::FRT-kan^(R)-FRT, pykF::FRT-kan^(R)-FRT, andcrr::FRT-kan^(R)-FRT deletion cassettes were individually PCR amplifiedfrom E. coli JW2580-1, JW1843-2, JW1666-3, and JW2410-1, respectfully.Chromosomal integration of said cassettes and subsequent removal ofkan^(R) markers was achieved as previously described (Datsenko, K. A.,Wanner, B. L., 2000. et al., Proc Natl Acad Sci USA. 97, 6640-5; Pugh,et al., 2014. Process Biochemistry. 49, 1843-1850), resulting in theindividual construction of E. coli NST74 ΔpheA, E. coli NST74 ΔpheAΔpykA ΔpykF, and E. coli NST74 ΔpheA ΔpykA ΔpykF Δcrr.

For catechol biosynthesis, E. coli NST74 ΔpheA was co-transformed withthe following combinations of plasmids (note: pathway designationprovided in parentheses, see Table 1): pY3 and pTutA-pPh (CAT2),pSDC-PchB-EntC and pPh (CAT3), pUbiC-Kpd and pPh (CAT4), and pUbiC-PobAand pAroY (CAT5). For MA biosynthesis, E. coli NST74 ΔpheA, NST74 ΔpheAΔpykA ΔpykF, and NST74 ΔpheA ΔpykA ΔpykF Δcrr were each co-transformedwith the following combinations of plasmids: pQsuB-AroY-CatA (MA1), pY3and pTutA-pPh-CatA (MA2), pSDC-PchB-EntC and pPh-CatA (MA3), pUbiC-Kpdand pPh-CatA (MA4), pUbiC-PobA and pAroY-CatA (MA5), and pUbiC-PobA andpQsuB-AroY-CatA (MAF).

Thermodynamic and Elementary Flux Mode (EFM) Analysis

To compare relative pathway energetics, net changes in Gibbs free energydue to reaction, Δ_(r)G′°_(net), were calculated using eQuilibrator(http://equilibrator.weizmann.ac.il) at a reference state of 25° C., pH7, and ionic strength of 0.1 M. Elementary flux modes (EFMs) werecomputed in MATLAB R2014b (MathWorks, Natick Mass.) using EFMTool 4.7.1(Terzer, M., Stelling, J., 2008. Bioinformatics. 24, 2229-35). An E.coli stoichiometric network originally employed to compare differentphenol biosynthesis pathways (Thompson, et al., 2016. Biotechnol Bioeng.113(8), 1745-54) and originally adapted from Averesch and Krömer(Metabolic Engineering Communications. 1, 19-28 (2014)), was used tocompare relative maximum yields from the various MA pathways and strainsof interest in this study.

Assaying Phenol Hydroxylase and Catechol 1,2-Dioxygenase Activity UsingWhole Resting Cells

Recombinant activities of phenol hydroxylase and catechol1,2-dioxygenase were assayed in E. coli BW25113 following itstransformation with pPh or pPh-CatA. Overnight seed cultures were usedto inoculate (1% vol.) 50 mL of LB supplemented with 20 g/L glucose and35 mg/L kanamycin in 250 mL shake flasks. In addition, the effect ofFe(NH₄)₂(SO₄)₂ was also examined via its omission or inclusion at 100μM. Shake flasks were induced by addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mMupon reaching an optical density at 600 nm (OD₆₀₀) of 0.7. Culturingcontinued overnight (˜12 h) at 32° C. before cells were then collectedby centrifugation at 3,000×g for 5 min. Cell pellets were rinsed twicewith pH 6.8 phosphate buffered saline (PBS) before being re-suspended toa final OD₆₀₀ of 4 in PBS supplemented with 0.2 g/L glucose and 1 mMphenol in a 250 mL shake flask. Cultures were subsequently incubated at32° C. with shaking at 200 RPM for up to 12 hours. Samples wereperiodically drawn for metabolite quantification via HPLC, as describedbelow.

Catechol and MA Production from Glucose

To investigate the bioproduction of catechol and MA, overnight seedcultures were first prepared in LB and supplemented with 100 mg/Lampicillin and/or 35 mg/L kanamycin and used to inoculate (1% vol) 50 mLof M9M minimal media supplemented with 20 g/L glucose in 250 mL shakeflasks (note: the medium was additionally supplemented with 0.1 g/L Phewhen using ΔpheA-derived host strains). Shake flask cultures wereincubated at 32° C. with shaking at 200 RPM until reaching OD₆₀₀˜0.7, atwhich point IPTG induction was performed at a final concentration of 0.4mM. Cultures were further incubated for a total of up to 120 h, or untilsignificant sugar consumption was no longer detected. Periodically,samples were drawn to measure cell growth (as OD₆₀₀) sugar andmetabolite levels by HPLC analysis, as described below. Prior tocentrifugation (i.e., to pellet and remove cells), samples for MAanalysis were first diluted 1:10 with methanol, while samples for Tyranalysis were diluted 1:10 with 1 N HCl and incubated at 55° C. for 30min. All samples were then centrifuged at 11,000×g for 5 min beforetransferring the supernatant to a glass HPLC vial.

HPLC Metabolite Analysis

Metabolite analysis was performed using a Hewlett Packard 1100 seriesHPLC system. Separation of Phe, pHBA, PCA, salicylate, phenol, catechol,and MA, was achieved using a reverse-phase Hypersil GOLD aQ C18 column(3 mm×250 mm; Thermo Fisher, Waltham, Mass., USA) operated at 45° C.with an isocratic 0.8 mL/min mobile phase consisting of 85% (vol.) 5 mMH2504 and 15% (vol.) acetonitrile. The eluent was monitored using adiode array detector (DAD) set at 215 nm for Phe, salicylate, PCA, andcatechol, 260 nm for pHBA, and 275 nm for phenol and MA. Separation ofTyr was also achieved on Hypersil GOLD aQ C18 column, in this casemaintained at 30° C. while using a mobile phase consisting of water (A)and methanol plus 0.1% (vol.) formic acid (B) at a constant flow rate of0.2 mL/min and the following concentration gradient (all by vol.): 5% Bfrom 0 to 8 min, 5% to 40% B from 8 to 13 min, 40% B from 13 to 16 min,40 to 5% B from 16 to 21 min, and 5% B from 21 to 31 min. The eluent wasmonitored using a DAD at 215 nm. Glucose and acetate separation wasachieved using an Aminex HPX-87H column (BioRAD, Hercules, Calif.)operated at 35° C. and detected using a refractive index detector (RID).The column was eluted with 5 mM H₂SO₄ at a constant flow rate of 0.55mL/min. In all cases, external standards were prepared and used toprovide calibrations for concentration determination.

Mass spectrometry analysis was performed using a Dionex Ultimate 3000HPLC system (Bruker Daltonics, Billerica, Mass., USA) consisting of aHPG-3400 M pump, WPS 3000 TB autosampler fitted with a 5 μL sample loop,and a FLM-3100B thermostatted column compartment. A Hypersil GOLD aQ C18column (3 mm×250 mm; Thermo Fisher, Waltham, Mass., USA) was operated at25° C. with an isocratic 0.2 mL/min mobile phase consisting of 85%(vol.) 5 mM formic acid and 15% (vol.) acetonitrile. Metabolites weredetected as negative ions using a Bruker MicrOTOF-Q mass spectrometerwith the following settings: Capillary voltage, +4000; end plate offset,−500V; nebulizer gas pressure, 2.0 bar; dry gas flow, 8 L/min; dry gastemperature, 210° C.; Funnels 1 and 2 radio frequency (RF) settings, 200Vpp, Hexapole RF setting, 150 Vpp; Collision Cell RF setting, 100 Vpp;Quadrupole low mass setting, 55 m/z; Transfer time, 100 μs; Pre PulseStorage, 7 μs. A Peak Scientific NM3OLA nitrogen generator (PeakScientific Inc., Billerica, Mass., USA) supplied nitrogen for the dryingand nebulizer gases.

Results and Discussion Novel Pathway Identification and TheoreticalComparison

In addition to the original, ‘3DHS-derived’ pathway (steps A, B, C inFIG. 1; hereafter referred to as MA1) (Draths, K. M., Frost, J. W.,1994. et al., Journal of the American Chemical Society. 116, 399-400),as discussed above, several alternative pathway options for MAbiosynthesis have since also been developed. Although proceeding fromdifferent endogenous metabolites and/or involving alternative enzymechemistries, catechol always serves as the immediate MA precursor.However, in addition to serving as the ubiquitous MA precursor, catecholis also the first intermediate associated with aerobic phenolcatabolism—a degradation pathway common to many soil microbes, includingvarious Pseudomonas sp., for example (van Schie, P. M., Young, L. Y.,2000. Bioremediation Journal. 4, 1-18). Accordingly, it was hypothesizedthat, by linking engineered phenol production with its partial, aerobicdegradation, additional new routes to MA could ultimately also beengineered. More specifically, as illustrated in FIG. 1, this could beachieved by further co-expressing phenol hydroxylase (PH) and catechol1,2-dioxygnease (CDO) (steps K, C; FIG. 1) in a phenol-producingbackground. Whereas engineered phenol production has traditionallyinvolved expression of tyrosine phenol lyase (TPL; step J) in a Tyroverproducing host (Kim, et al., 2014. Biotechnology journal. 9, 621-9;Wierckx, et al., 2005. Applied and environmental microbiology. 71,8221-7), the engineering of two alternative phenol biosynthesis pathwaysfrom chorismate was recently reported, involving either isochorismatesynthase, isochorismate pyruvate lyase, and salicylate decarboxylase(steps G, H, I) or chorismate lyase and pHBA decarboxylase (steps D, F)(Thompson, et al., 2016. Biotechnol Bioeng. 113(8), 1745-54).Accordingly, three novel, phenol-dependent MA biosynthesis pathways wereproposed, hereafter referred to as MA2 (steps J, K, C), MA3 (steps G, H,I, K, C), and MA4 (steps D, F, K, C). Meanwhile, in separate works,reported a novel pathway for catechol biosynthesis that proceeds fromchorismate through pHBA and PCA via chorismate lyase, pHBA hydroxylase,and PCA decarboxylase (steps D, E, B) was also previously reported(Pugh, et al., 2014. Process Biochemistry. 49, 1843-1850). Accordingly,further extension of this pathway to MA was also proposed, in this caseby co-expressing CDO, resulting in pathway MA5 (steps D, E, B, C).

Unlike MA1, the four alternative pathways proposed herein (MA2-5)importantly stem from chorismate or one of its downstream metabolites,and thereby offer improved host compatibility by preserving native fluxthrough the shikimic acid pathway. In addition, relative to MA1, each ofMA2-5 was also found to further benefit from an increased netthermodynamic driving force (as much as 104% greater). Morespecifically, when evaluated from the last common precursor (i.e.,3DHS), the net change in Gibbs free energy due to reaction(Δ_(r)G′°_(net)) was predicted to be −1037.4 kJ/mol for each of MA3-5and −1007.6 kJ/mol for MA2, compared to just −508.5 kJ/mol for MA1 (FIG.2). In contrast, as determined via elementary mode analysis, MA1supports the highest product yield, with the predicted maximumtheoretical yields of MA from glucose (Y_(P/S,max)) being ˜18% lower (or˜15% lower with growth, Y_(P/S,max+growth); FIG. 2) for each of MA2-5.In all cases, said reduction is due to the additional 1 NADPH (by AroE),1 ATP (by AroL), and 1 phosphoenolpyruvate (PEP; by AroA) consumed whileconverting 3DHS to chorismate. Meanwhile, both MA3 and MA4 furtherrequire an additional 1 NADH (i.e., by PH; note: this demand is balancedin MA2 by the generation of 1 NADH by TyrA), whereas MA5 requires anadditional 1 NADPH (by pHBA hydroxylase). Other previously-reportedchorismate-derived MA pathways (i.e., derived from anthranilate or fromsalicylate, via an alternative mechanism (Lin, et al., 2014. Metabolicengineering. 23; Sun, et al., 2013a. Applied and environmentalmicrobiology. 79)) suffer the same limitations with respect topredicated maximum MA yields from glucose, in this case also being about17-20% lower than MA1 (or 18-19% lower with growth; note: theanthranilate-derived pathway offers the lowest yield due to arequirement for an additional 1 ATP) (Averesch, et al., 2014. MetabolicEngineering Communications. 1, 19-28). Therefore, despite incorporatingmore favorable enzyme chemistries and the potential for improved hostcompatibility, potential improvements associated with the proposed andother MA pathways come at the cost of lower maximum theoretical yields.

Screening for and Characterizing Recombinant Phenol Hydroxylase Activityin E. coli

Effective enzyme candidates for most steps associated with each of theproposed pathways have been reported and/or characterized (Lin, et al.,2014. Metabolic engineering. 23; Pugh, et al., 2014. ProcessBiochemistry. 49, 1843-1850; Thompson, et al., 2016. Biotechnol Bioeng.113(8), 1745-54). Phenol-dependent MA biosynthesis (i.e. via pathwaysMA2-4) represents a new approach, however, and first requiredidentification of a candidate gene whose expression would conferrecombinant PH activity in E. coli. PH activity has been identified aspart of bacterial multicomponent monooxygenases (BMMs). BMMs represent abroad class of enzymes capable of using molecular oxygen to hydroxylatevarious hydrocarbon species, and have been identified to function innumerous microbes as the initial activating steps involved in degradingbenzene, toluene, and xylene (Jindrova, et al., 2002. FoliaMicrobiologica. 47, 83-93; Notomista, et al., 2003. J Mol Evol. 56,435-45; Sridevi, et al., 2012. Int J Eng Sci Adv Technol. 2, 695-705).Notable examples include toluene/o-xylene monooxygenase (ToMo, encodedby touABCDEF) and phenol hydroxylase (PH, encoded by phKLMNOP), bothfrom Pseudomonas stutzeri OX1, which together function to sequentiallycatalyze the first two steps in benzene degradation (i.e., via phenolthen catechol) (Cafaro, et al., 2004. Applied and environmentalmicrobiology. 70, 2211-9; Cafaro, et al., 2002. Eur J Biochem. 269,5689-99; Tinberg, et al., 2011. Biochemistry. 50, 1788-1798). Aspromiscuous enzymes, previous in vitro studies have shown that bothtouABCDEF and phKLMNOP display the desired PH activity; however,phKLMNOP exhibits more favorable activity towards phenol (K_(m)=0.6 μM,k_(cat)=1.02 s⁻¹), its native substrate, than does touABCDEF (K_(m)=2.18μM, k_(cat)=1.00 s⁻¹) (Cafaro, et al., 2004. Applied and environmentalmicrobiology. 70, 2211-9). Accordingly, phKLMNOP was selected as theinitial candidate for establishing recombinant PH activity (step K;FIG. 1) in E. coli. Meanwhile, whereas CDO activity (step C) has beenidentified in a variety of aromatic-degrading microorganisms (Bouwer, E.J., Zehnder, A. J., 1993. Trends in biotechnology. 11, 360-7; Cao, etal., 2008. Applied microbiology and biotechnology. 81, 99-107; Kukor, etal., 1988. Journal of Bacteriology. 170, 4458-4465), CatA from P. putidadisplays high recombinant activity (10.10±0.35 μmol/min/mg protein)(Sun, et al., 2013a. Applied and environmental microbiology. 79), andthus was accordingly used in each of MA1-5.

Recombinant PH activity was confirmed in vivo via whole resting cellassays employing E. coli BW25113 pPh. Because PH possessescarboxylate-bridged diiron catalytic centers in each of its N subunits,as well as a [2Fe-2S] cluster in the P subunit (Cafaro, et al., 2004.Applied and environmental microbiology. 70, 2211-9; Tinberg, et al.,2011. Biochemistry. 50, 1788-1798), the medium was first supplementedwith 100 μM Fe(NH₄)₂(SO₄)₂. Under these conditions, 1 mM exogenousphenol was rapidly converted to catechol, achieving a maximum specificrate of 0.991 mmol g⁻¹ h⁻¹ ⁽ FIG. 3A). However, after reaching a maximumlevel of 0.83±0.04 mM after 40 min, catechol levels then graduallydecreased over time, ultimately being undetected after 8 h. It wasinitially hypothesized that the disappearance of catechol was a resultof dimer formation, as said phenomena have been reported to occur forvarious catechols in the presence of excess Fe(III) and under mildlyacidic conditions, a process that plays an important role in adhesiveformation in the mussel byssus (Fullenkamp, et al., 2014. RSC Advances.4, 25127-25134). To test this hypothesis, and determine if such anundesirable side reaction could perhaps be avoided, the assay was nextrepeated without Fe(NH₄)₂(SO₄)₂ supplementation (FIG. 3A). Under theseconditions, phenol conversion to catechol occurred at a similar yield(0.85±0.03 mM catechol produced from 1 mM phenol after 1.5 h),proceeding, however, at only ˜60% of the previous maximum specific rate(just 0.596 mmol g⁻¹ h⁻¹). However, even in the absence of Fe(III),catechol was again depleted following its formation, here completelydisappearing after 12 h. As an alternative hypothesis, several BMMs havebeen reported to display promiscuous activity towards numerous, relatedaromatic species, including the repeated hydroxylation of the sameinitial substrate molecule (Tao, et al., 2004. Applied and environmentalmicrobiology. 70, 3814-20; Vardar, G., Wood, T. K., 2005. Journal ofBacteriology. 187, 1511-1514). Thus, it was postulated that the PHencoded by phKLMNOP might also be capable of further hydroxylatingcatechol to 1,2,3-trihydroxybenzene (1,2,3-THB). LC-MS analysis wasperformed on supernatants prepared from the above resting cell assays,wherein an unknown peak was identified with a molar mass (m/z) of125.03±0.02 (FIG. 4). As the molecular weight of 1,2,3-THB 126.11 g/mol,this strongly suggests that catechol disappearance is occurring due toits further hydroxylation to 1,2,3-THB by PH. Although previouslyreported for ToMo (Vardar, G., Wood, T. K., 2005. Journal ofBacteriology. 187, 1511-1514), to the best of the inventors' knowledge,this is the first report of such behavior by phKLMNOP from P. stutzeriOX1.

For any pathway incorporating PH (i.e., MA2-4), co-production of1,2,3-THB will likely compete for available catechol, thereby reducingMA production. Accordingly, it was hypothesized that rapid turnover ofcatechol to MA by CDO might enable the undesirable production of1,2,3-THB to be minimized upon implementation of the full pathway. As apreliminary test, the above experiment was repeated using whole restingcells of E. coli BW25113 pPh-CatA which co-express CDO (encoded by catAfrom P. putida KT2440) together with PH. In this case, as seen in FIG.3B for both with and without Fe(NH₄)₂(SO₄)₂ supplementation, 1 mM phenolwas converted to MA at maximum final concentrations of 0.62±0.08 and0.12±0.01 mM, respectively. Thus, although co-expression of PH and CDOenabled the successful transformation of phenol to MA (which, unlikecatechol, stably accumulated for the duration of the experiment; FIG.3B), overall conversion was low (reaching no greater than 62%) due tothe persistent competing formation of 1,2,3-THB by PH.

Investigating Phenol-Dependent Catechol Production in E. coli

Three distinct phenol biosynthesis pathways (Thompson, et al., 2016.Biotechnol Bioeng. 113(8), 1745-54) were each next extended to catecholvia the additional co-expression of phKLMNOP. This resulted in theconstruction of three novel catechol production pathways: CAT2 (steps J,K), CAT3 (steps G, H, I, K), and CAT4 (steps D, F, K) (FIG. 1). Whenexpressed in E. coli NST74 ΔpheA in shake flask cultures,phenol-dependent catechol biosynthesis from glucose was successfullydemonstrated in all three cases, the results of which are compared inTable 3. For each of CAT2-4, however, significant levels of residualphenol remained at the end of each culture (reaching as high as 149±1mg/L and, in all cases, surpassing total catechol production),suggesting low PH activity as a common bottleneck. In addition, similarto the whole resting cell assays, all three pathways further sufferedfrom the competing formation of 1,2,3-THB, with detected levels reachingas much as 96±4 mg/L. In the case of CAT2, catechol titers reached 79±3mg/L at a glucose yield of 6.2±0.28 mg/g. In addition to phenol, highlevels of unconverted Tyr also accumulated in the medium, reaching342±12 mg/L after 96 h, pointing to TPL as an additional flux limitingstep in the pathway. TPL, for example, is known to suffer from bothequilibrium limitations (the reaction is reversible, with Δ_(r)G′°=+27.9kJ/mol; FIG. 1) as well as feedback inhibition by phenol (e.g., 94 mg/Lphenol reduces TPL activity to only 23% of its maximum (Wierckx, et al.,2008. Journal of bacteriology. 190, 2822-30)). Among the three pathways,CAT3 enabled the highest catechol titer and yield, reaching 100±2 mg/Land 12.8±0.26 mg/g, respectively; however, residual salicylate alsoaccumulated at 55±2 mg/L in this case. Moreover, glucose utilizationremained low among all strains, with overall consumption averaging just˜44% (Table 3), the precise reasons for which presently remain unclear.Thus, although functional and enabling phenol-dependent catecholproduction from glucose for the first time, all three novel catecholpathways (i.e., CAT2-4) suffer from inherent limitations associated withPH, both in terms of low recombinant activity as well as its apparentpromiscuity. For comparison, the focal production of catechol viaanother, chorismate-derived pathway, referred to here as CAT5 (steps D,E, B; FIG. 1) was previously investigated (Pugh, et al., 2014. ProcessBiochemistry. 49, 1843-1850). When expressed in the same host backgroundand under identical culture conditions, said pathway enabled finalcatechol titers reaching up to 451±44 mg/L at a glucose yield of35.0±3.0 mg/g—outputs 4.5- and 2.7-fold greater, respectively, thanthose achieved by even CAT3—and did so without significant terminalaccumulation of any pathway intermediates.

Investigating MA Production Via Newly Engineered Pathways

The relative prospects of MA biosynthesis via the three phenol-dependentpathways (MA2-4) and the previously-reported, chorismate-derivedcatechol pathway (MA5) were investigated via the additionalco-expression of catA from P. putida KT2440 (encoding CDO). E. coliNST74 ΔpheA was again employed as the initial host background ofinterest, and the results are compared in Table 4. MA titers by MA2,which proceeds through phenol via Tyr, were lowest, reaching just 186±11mg/L at a glucose yield of 21.0±2.2 mg/g. As was the case for CAT2, thisappears to be due at least in part to flux limitations associated withboth TPL and PH, as indicated by the terminal accumulation of as much as220±12 mg/L Tyr and 63±1 mg/L phenol. Like CAT3, MA3 resulted in thehighest MA titers and yields among all phenol-derived pathways (i.e.,MA2-4), reaching 484±44 mg/L and 46.7±6.0 mg/g, respectively. However,analogous to the case of catechol production, the common reliance uponPH in each of MA2-4 similarly resulted in production of 1,2,3-THBbyproduct in each case, here reaching as high as 232±17 mg/L.Furthermore, similar to CAT2-4, low glucose consumption was alsoobserved for each of MA2-4, again averaging just 44% (Table 4).Meanwhile, perhaps expectedly, MA biosynthesis was highest in the caseof MA5, reaching 819±44 mg/L at a yield of 40.9±2.2 mg/g. Lastly, toprovide a head-to-head comparison, MA1 (i.e., the original‘3DHS-derived’ pathway) was also constructed and expressed in the samehost background. In this case, MA production reached 1586±11 mg/L at ayield of 79.3±0.53 mg/g. Overall, said results are consistent with theabove model predictions that found MA1 to be the highest yield pathway(FIG. 2). Interestingly, and in contrast to the original Draths andFrost study wherein an E. coli ΔaroE background was used to promote 3DHSavailability (resulting in multiple, undesirable auxotrophies),significant MA production via MA1 was demonstrated here withoutdisrupting the shikimic acid pathway. This suggests that, at least underthe present conditions, MA1 is capable of effectively competing againstnative metabolism for at least significant portion of available 3DHS.Furthermore, it should also be noted that although low PCA decarboxylaseactivity has been implicated as a limiting factor associated with MAproduction via MA1 (and here, by extension, perhaps also MA5) (Sonoki,et al., 2014. J Biotechnol. 192 Pt A, 71-7), as PCA remained undetectedthroughout any of the present cultures (Table 4), such effects do notappear to be limiting for the strains and conditions studied here.

Enhancing MA Production Via Synthetic ‘Metabolic Funneling’

To further improve upon the high production potential of MA1, asynthetic ‘metabolic funnel’ was next engineered and investigated as anovel strategy for further improving MA biosynthesis. More specifically,an additional ‘funneling’ pathway, referred to as MAF (steps A, B, C, D,E; FIG. 1), was engineered in this case by co-expressing both MA1 andMA5 together in the same host (FIG. 5). In this way, MA productionoccurs via two simultaneous and compatible routes: first, via the highyield, ‘3DHS-derived’ pathway (MA1) and, second, via thebest-performing, chorismate-derived pathway (MA5)—the latter of whichprovides an opportunity to ‘rescue’ additional endogenous precursors notinitially ‘captured’ at first branch point (i.e., at 3DHS). Accordingly,with two routes functioning in parallel, it was proposed that MAF wouldenable maximal total precursor assimilation towards MA. As withindividual MA pathways, MAF was first introduced and expressed in NST74ΔpheA, resulting in the accumulation of 2042±88 mg/L MA at a glucoseyield of 102±4.4 mg/g (Table 4). When compared to MA1, this suggeststhat as much as an additional 456 mg/L MA (22% of total MA) was producedvia the ability of the ‘lower branch’ (i.e., MA5) to assimilateprecursors (in this case, chorismate) not initially taken in via the‘upper branch’ (i.e., MA1). Overall, when compared to strains expressingthe single, parent pathways (i.e., MA1 or MA5 alone), the combined‘funneling’ strategy of MAF resulted in MA titer improvements of 29 and150%, respectively, and 29 and 132% in terms of achievable yields.Finally, in contrast to each of the phenol-derived pathways (i.e.,MA2-4), when employing MA1, MA5, or MAF, all available glucose wasconsumed within 120 h (Table 4).

Host Engineering to Enhance Precursor Availability and MA Production

To further improve MA production, subsequent culturing and strainengineering efforts were focused on increasing total carbon flux intothe shikimic acid pathway and reducing overflow metabolism observed withhigh glucose uptake rates (Liu, et al., 2014. Process Biochemistry. 49,751-757). Flux into the shikimic acid pathway is initially controlled by3-deoxy D-arabinoheptulose 7-phosphate (DAHP) synthase, whose twosubstrates are phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P)(Bongaerts, et al., 2001. Metabolic engineering. 3; Gosset, G., 2009.Current opinion in biotechnology. 20; Rodriguez, et al., 2014. MicrobCell Fact. 13). As has been previously demonstrated (Gosset, G., 2005.Microb Cell Fact. 4, 14; Postma, et al., 1993. Microbiol Rev. 57),increasing the intracellular availability of PEP is an effectivestrategy for enhancing the production of aromatic amino acids (Liu, etal., 2014. Process Biochemistry. 49, 751-757) and other products fromintermediates of the shikimic acid pathway (Noda, et al., 2016.Metabolic engineering. 33, 119-129). In glucose-fed cultures, PEPavailability can be increased by blocking its conversion to pyruvate viadeletion of pykA and pykF, both of which encode isozymes of pyruvatekinase. Meanwhile, rapid uptake of glucose has been previously reportedto result in the accumulation of acetate (Gosset, G., 2005. Microb CellFact. 4, 14)—an undesirable byproduct which can ultimately inhibit cellmetabolism (Shiloach, et al., 1996. Biotechnol Bioeng. 49, 421-8 et al.,1996) and result in lower aromatic product yields (Liu, et al., 2014.Process Biochemistry. 49, 751-757). As seen in Table 4, for example,significant levels of residual acetate were observed here in all cases,reaching as high as 12 g/L. It has previously been shown, however, thatcarbohydrate repression resistant null mutants (i.e., Δcrr) displaylower rates of glucose uptake and thus reduced overflow metabolism. As aresult, this mutation has also been previously demonstrated as effectivefor enhancing phenylalanine production (Liu, et al., 2014. ProcessBiochemistry. 49, 751-757). Accordingly, E coli NST74 ΔpheA ΔpykA ΔpykFΔcrr was next constructed and evaluated as a MA production host, in thiscase narrowing the focus to just MA1 and MAF. As seen in Table 5, forMA1, whereas acetate accumulation was effectively eliminated, theadditional mutations enabled a modest (˜13%) increase in MA production,with final titers reaching 1792±28 mg/L at a glucose yield of 89.6±1.4mg/g. On the other hand, MA production by MAF was significantly enhancedusing E coli NST74 ΔpheA ΔpykA ΔpykF Δcrr as host, with final MA titersreaching 3153±149 mg/L at a glucose yield of 158±7.4 mg/g—both increaseof over 1.5-fold relative to the use of E coli NST74 ΔpheA as host, andthe highest production metrics achieved in this study. Furthermore, thismaximal titer also is 31% higher than the 2.4 g/L of MA reported byDraths and Frost via MA1 in an E. coli ΔaroE background (Draths, K. M.,Frost, J. W., 1994. et al., Journal of the American Chemical Society.116, 399-400), and was notably achieved while generating only a singleauxotrophy (i.e., for Phe).

Further comparing the results of Tables 4 and 5, although the apparentflux of precursor through the ‘upper branch’ (i.e., MA1) was onlyslightly improved, the ‘lower branch’ (i.e., MA5) appeared to offer aneven greater benefit; in this case enabling the production of as much asan additional 1361 mg/L MA (43% of total MA produced). Accordingly, theadditional ΔpykA, ΔpykF, and Δcrr mutations appear to have more greatlyimproved the intracellular availability of chorismate than that of 3DHS.Interestingly, meanwhile, in the case of MAF, acetate accumulationreemerged, reaching up to 7±0.1 g/L by the end of the culture (Table 5).Unlike MA1, the first step of MA5 (step D; FIG. 1) is catalyzed bychorismate pyruvate lyase (encoded by native ubiC) and results in thegeneration of 1 pyruvate molecule. While future works will be requiredto test this hypothesis, it is possible that said pyruvate is thenconverted to acetyl-CoA and ultimately to acetate via E. coli'sphosphate acetyltransferase and acetate/acetyl-CoA pathway (encoded bypta-ackA), providing the cell with 1 ATP in the process. Further hostengineering to incorporate additional mutations previously reported aseffective for reducing acetate accumulation during aromatic amino acidbiosynthesis may also be performed (Castaño-Cerezo, et al., 2009. etal., Microb Cell Fact. 8, 54; Liu, et al., 2016. Plos One. 11, e0158200;Wang, et al., 2013. Applied microbiology and biotechnology. 97, 7587-96;Wolfe, A. J., 2005. Microbiology and Molecular Biology Reviews. 69,12-50) with the goal of further improving MA production via thedemonstrating funneling pathway, MAF.

Example 2. Engineering Novel Pathways and a Synthetic Metabolic Funnelto Enhance Phenol Biosynthesis

Phenol is an important building block molecule used in the synthesis ofvarious specialty chemicals, plastics and polymers of industrialrelevance (Adkins, et al., 2012. Frontiers in microbiology. 3, 313;Deng, et al., 2016. Biochem Eng J. 105, 16-26). Although the completebiosynthesis of phenol from glucose has previously been demonstrated,the originally engineered pathway, which proceeds from endogenoustyrosine via tyrosine phenol lyase (TPL) (Wierckx, et al., 2008. Journalof bacteriology. 190, 2822-30; Wierckx, et al., 2005. Applied andenvironmental microbiology. 71, 8221-7), suffers from notable inherentlimitations. Phenol has previously been synthesized from endogenoustyrosine via expression of heterologous tyrosine phenol lyase (TPL)activity (FIG. 6). For instance, although phenol was successfullysynthesized in this manner with maximal shake flask titers reaching 141mg/L (Wierckx, et al., 2005. Applied and environmental microbiology. 71,8221-7), TPL reaction reversibility and enzymatic feedback inhibitionultimately resulted in poor pathway efficiency and limited productionmetrics (Kim, et al., 2014. Biotechnology journal. 9, 621-9; Wierckx, etal., 2008. Journal of bacteriology. 190, 2822-30). As an alternativeapproach, two novel pathways were engineered and comparatively evaluatedagainst the original tyrosine-derived pathway (FIG. 6), both of whichinstead stem from endogenous chorismate (Thompson, et al., 2016.Biotechnol Bioeng. 113(8), 1745-54). Using these pathways, titersreached as high as 377 mg/L, although overall yields via any phenolpathway remained less than 10% of the theoretical maximum (Thompson, etal., 2016. Biotechnol Bioeng. 113(8), 1745-54). In the present example,a synthetic ‘metabolic funnel’ was engineered and investigated as astrategy for enhancing phenol production via the parallel co-expressionof distinct yet converging biosynthesis pathways (FIG. 7).

Materials and Methods Strains and Media

All strains used in this study are listed in Table 6. E. coli NEB10-beta(New England Biolabs (NEB); Ipswich, Mass.) was used for all cloning andplasmid maintenance. E. coli NST74 (ATCC 31884) was obtained from theAmerican Type Culture Collection (ATCC; Manassas, Va.) and served as theparent strain in this study. E. coli JW2580-1, JW1843-2, JW1666-3, andJW2410-1 were obtained from the Coli Genetic Stock Center (CGSC; NewHaven, Conn.) and served as the genetic source for the pheA::Kan^(R),pykA::Kan^(R),pykF::Kan^(R), and crr::Kan^(R) cassettes, respectively.E. coli BW25113 was obtained from the CGSC and served as the geneticsource for ubiC and entC. Citrobacter braakii (ATCC 29063) was obtainedfrom the ATCC and served as the genetic source for tutA. Klebsiellapneumoniae PZH572 (ATCC 25955) was obtained from the ATCC and served asthe genetic source of kpdBCD. Pseudomonas aeruginosa PAO1 (DSMZ 22644)was obtained from the Leibniz Institute German Collection ofMicroorganisms and Cell Cultures and served as the genetic source ofpchB.

Seed cultures of E. coli strains were cultured in Luria-Bertani (LB)broth at 32° C. and supplemented with 100 mg/L ampicillin, 35 mg/Lkanamycin, and/or 34 mg/L chloramphenicol, as appropriate. For phenolbiosynthesis, shake flasks were cultured at 32° C. in MM1 phosphatelimited minimal media supplemented with appropriate antibiotics. MM1 wascomposed of the following (in g/L): MgSO₄.7H₂O (0.5), (NH₄)₂SO₄ (4.0),MOPS (24.7), KH₂PO₄ (0.3), K₂HPO₄ (0.7), and glucose (20). Traceelements were supplemented in MM1 as follows (in mg/L):(NH₄)₆Mo₇O₂₄.4H₂O (0.37), H₃BO₃(2.5), CoCl₂.6H₂O (0.714), CuSO₄ (0.16),MnCl₂.4H₂O (1.6), ZnSO₄.7H₂O (0.288), FeCl₃ (0.05).

Plasmid Construction

All plasmids used and developed in this study are listed in Table 6. Allgenes were PCR amplified with Q5 High-Fidelity DNA Polymerase (NEB) anda BioRad iCycler, per manufacturer protocols. Custom DNA oligonucleotideprimers (Table 7) were synthesized by Integrated DNA Technologies (IDT,Coralville Iowa). Genomic DNA (gDNA) templates were prepared using theZR Fungal/Bacterial DNA MiniPrep kit while plasmid DNA was purifiedusing the Zymo Plasmid MiniPrep kit (both Zymo Research, Irvine Calif.).Amplified linear DNA fragments were purified using the Zymo DNA Clean &Concentrator MiniPrep kit (Zymo Research). Select purified linear andplasmid DNA were digested using appropriate restriction endonucleases(NEB) and subsequently gel purified using the Zymoclean Gel DNA RecoveryMiniPrep kit (Zymo Research). Purified digested DNA fragments wereligated using T4 DNA Ligase (NEB), per manufacturer protocols.Alternatively, purified linear DNA was subsequently used as template DNAfor either circular polymerase extension cloning (CPEC) (Quan, J., Tian,J., 2011. Nat. Protocols. 6, 242-251) with Q5 High-Fidelity DNAPolymerase according to manufacturer protocols, or Gibson Assembly(Gibson, et al., 2009. Nature methods. 6, 343-5) using Gibson AssemblyMaster Mix (NEB) according to manufacturer protocols. Ligation, CPEC,and Gibson Assembly reactions were transformed into chemically competentE. coli NEB10-beta before plating on LB solid agar media supplementedwith appropriate antibiotics for selection. Transformant pools werescreened using colony PCR, restriction digest mapping, and finallyconfirmed by DNA sequencing.

Strain Construction

Chromosomal in-frame deletions of pheA in E. coli NST74 was constructedusing a modified version of the Datsenko and Wanner method (Datsenko, K.A., Wanner, B. L., 2000. et al., Proc Natl Acad Sci USA. 97, 6640-5), aspreviously described (Pugh, et al., 2014. Process Biochemistry. 49,1843-1850). The pheA::FRT-kan^(R)-FRT, pykA::FRT-kan^(R)-FRT,pykF::FRT-kan^(R)-FRT, and crr::FRT-kan^(R)-FRT deletion cassettes werePCR amplified from E. coli JW2580-1, JW1843-2, JW1666-3, and JW2410-1,respectfully. Chromosomal integration of said cassette and subsequentremoval of kan^(R) marker was achieved as previously described(Datsenko, K. A., Wanner, B. L., 2000. et al., Proc Natl Acad Sci USA.97, 6640-5; Pugh, et al., 2014. Process Biochemistry. 49, 1843-1850),resulting in the individual construction of E. coli NST74 ΔpheA and E.coli NST74 ΔpheA ΔpykA ΔpykF Δcrr.

For phenol biosynthesis, E. coli NST74 ΔpheA and E. coli NST74 ΔpheAΔpykA ΔpykF Δcrr was co-transformed with the following combinations ofplasmids (pathway designations provided in parentheses, see Table 8):pTyrAfbr-TutA (PH1); pSDC-PchB-EntC (PH2); pUbiC-Kpd (PH3);pTyrAfbr-TutA and pSDC-PchB-EntC (PHF1); pTyrAfbr-TutA and pUbiC-Kpd(PHF2); pSDC-PchB-EntC and pUbiC-Kpd (PHF3); pTyrAfbr-TutA,pSDC-PchB-EntC, and pUbiC-Kpd (PHF4).

Phenol Bioproduction

To investigate the bioproduction of phenol, overnight seed cultures werefirst prepared in LB and supplemented with 100 mg/L ampicillin, 35 mg/Lkanamycin, and/or 34 mg/L chloramphenicol and used to inoculate (1% vol)50 mL of MM1 minimal media supplemented with 20 g/L glucose in 250 mLshake flasks (note: the medium was additionally supplemented with 0.1g/L Phe when using ΔpheA-derived host strains). Shake flask cultureswere incubated at 32° C. with shaking at 200 RPM until reachingOD₆₀₀˜0.7, at which point IPTG induction was performed at a finalconcentration of 0.4 mM. Cultures were further incubated for a total ofup to 120 h, or until significant sugar consumption was no longerdetected. Periodically, samples were drawn to measure cell growth (asOD₆₀₀) as well as sugar and metabolite levels by HPLC analysis, asdescribed below. Prior to centrifugation, samples for Tyr analysis werediluted 1:10 with 1 N HCl and incubated at 55° C. for 30 min. Allsamples were then centrifuged at 11,000×g for 5 min before transferringthe supernatant to a glass HPLC vial.

HPLC Metabolite Analysis

Metabolite analysis was performed using a Hewlett Packard 1100 seriesHPLC system. Separation of Phe, pHBA, salicylate, and phenol wasachieved using a reverse-phase Hypersil GOLD aQ C18 column (3 mm×250 mm;Thermo Fisher, Waltham, Mass., USA) operated at 45° C. with an isocratic0.8 mL/min mobile phase consisting of 85% (vol.) 5 mM H2504 and 15%(vol.) acetonitrile. The eluent was monitored using a diode arraydetector (DAD) set at 215 nm for salicylate, 260 nm for pHBA, and 275 nmfor phenol. Separation of Tyr was also achieved on the same HypersilGOLD aQ C18 column, in this case maintained at 30° C. while using amobile phase consisting of water (A) and methanol plus 0.1% (vol.)formic acid (B) at a constant flow rate of 0.2 mL/min and the followingconcentration gradient (all by vol.): 5% B from 0 to 8 min, 5% to 40% Bfrom 8 to 13 min, 40% B from 13 to 16 min, 40 to 5% B from 16 to 21 min,and 5% B from 21 to 31 min. The eluent was monitored using a DAD at 215nm. Glucose and acetate separation was achieved using an Aminex HPX-87Hcolumn (BioRad, Hercules, Calif.) operated at 35° C. and detected usinga refractive index detector (RID). The column was eluted with 5 mM H₂SO₄at a constant flow rate of 0.55 mL/min. In all cases, external standardswere prepared and used to provide calibrations for concentrationdetermination.

Results and Discussion Demonstrating Phenol and MA Production ViaSynthetic ‘Metabolic Funneling’

A synthetic ‘metabolic funneling’ approach was investigated as a novelstrategy for improving phenol bioproduction metrics. Multiple‘funneling’ strategies were investigated by co-expressing both theoriginal tyrosine-derived pathway (PH1, FIG. 7) with the salicylatederived pathway (PH2, FIG. 7) and/or the p-hydroxybenzoate (pHBA)derived pathway (PH3, FIG. 7). In this way, phenol production occurs viaeither two or three simultaneous and compatible routes (PHF1-4, Table8). Phenol ‘funneling’ pathways were first introduced and expressed inE. coli NST74 ΔpheA. In the case of PHF1, titers reached 439±7 mg/L (a16% increase over the best-performing, single pathway control) at aglucose yield of 24.0±0.51 mg/g. In the case of PHF2, titers reached355±17 mg/L at a glucose yield of 17.8±1.2 mg/g. Meanwhile, phenoltiters by PHF3 reached just 149±10 mg/L at a glucose yield of 8.4±1.0mg/g. Finally, with all three pathways expressed simultaneously (PHF4),phenol titers reached 205±13 mg/L at a glucose yield of 11.8±0.72 mg/g.Meanwhile, significant acetate accumulation was observed in all cases(Table 9) suggesting high rates of glucose consumption and significantoverflow metabolism.

Host Engineering to Enhance Precursor Availability

To further improve phenol production, subsequent strain engineeringefforts were focused on increasing total carbon flux into the shikimicacid pathway and reducing overflow metabolism observed with high glucoseuptake rates (Liu, et al., 2014. Process Biochemistry. 49, 751-757).Flux into the shikimic acid pathway is initially controlled by 3-deoxyD-arabinoheptulose 7-phosphate (DAHP) synthase, whose two substrates arephosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) (Bongaerts, etal., 2001. Metabolic engineering. 3; Gosset, G., 2009. Current opinionin biotechnology. 20; Rodriguez, et al., 2014. Microb Cell Fact. 13). Aspreviously demonstrated (Gosset, G., 2005. Microb Cell Fact. 4, 14;Postma, et al., 1993. Microbiol Rev. 57), increasing the intracellularavailability of PEP is an effective strategy for enhancing theproduction of aromatic amino acids (Liu, et al., 2014. ProcessBiochemistry. 49, 751-757) and other products from intermediates of theshikimic acid pathway (Noda, et al., 2016. Metabolic engineering. 33,119-129). In glucose-fed cultures, PEP availability can be increased byblocking its conversion to pyruvate via deletion of pykA and pykF, bothof which encode isozymes of pyruvate kinase. Meanwhile, rapid uptake ofglucose has been previously reported to result in the accumulation ofacetate (Gosset, G., 2005. Microb Cell Fact. 4, 14)—an undesirablebyproduct which can ultimately inhibit cell metabolism (Shiloach, etal., 1996. Biotechnol Bioeng. 49, 421-8) and result in lower aromaticproduct yields (Liu, et al., 2014. Process Biochemistry. 49, 751-757).Carbohydrate repression resistant null mutants (i.e., Δcrr) have beenpreviously shown to display lower rates of glucose uptake and thusreduced overflow metabolism—a strategy demonstrated as effective forenhancing phenylalanine production (Liu, et al., 2014. ProcessBiochemistry. 49, 751-757). Accordingly, E coli NST74 ΔpheA ΔpykA ΔpykFΔcrr was next constructed and evaluated as a phenol production host.

Here, the focus was narrowed to the three best performing pathways: PH1,PH3, and PHF1. The individual pathways PH1 and PH3 displayed a modestdecrease in titer and yield when using E coli NST74 ΔpheA ΔpykA ΔpykFΔcrr as host, with final phenol titers reaching 329±9 and 277±15 mg/L,respectively (Table 10). On the other hand, phenol production by PHF1was slightly enhanced, with final titers reaching 575±19 at a glucoseyield of 28.8±0.34—a 1.3 and 1.2-fold increase relative to the previousgeneration strain. In addition, the titer demonstrated by PHF1represents a 4-fold increase over the original phenol biosynthesisreported using the solvent tolerant P. putida (Wierckx, et al., 2005.Applied and environmental microbiology. 71, 8221-7) and a 1.4-foldincrease over the highest E. coli derived phenol production reported todate (Kim, et al., 2014. Biotechnology journal. 9, 621-9).

Tables

TABLE 1 Strains, plasmids, and pathways constructed and/or used inExample 1. Strain Description Source E. coli NEB 10-beta Δ(ara-leu) 7697araD139 fhuA ΔlacX74 galK16 galE15 NEB e14- ϕ80dlacZΔM15 recA1 relA1endA1 nupG rpsL (Str^(R)) rph spoT1 Δ(mrr-hsdRMS-mcrBC) E. coli BW25113Source of ubiC and entC CGSC E. coli JW2580-1 Source of pheA::Kan^(R)CGSC E. coli JW1843-2 Source of pykA::Kan^(R) CGSC E. coli JW1666-3Source of pykF::Kan^(R) CGSC E. coli JW2410-1 Source of crr::Kan^(R)CGSC E. coli NST74 aroH367, tyrR366, tna-2, lacY5, aroF394(fbr),malT384, ATCC pheA101(fbr), pheO352, aroG397(fbr) E. coli NST74pheA^(fbr) chromosomal deletion in E. coli NST74 Pugh et al. ΔpheA^(fbr)(2014) C. glutamicum Source of qsuB ATCC C. braakii Source of tutA ATCCP. aeruginosa PAO1 Source of pobA and pchB DSMZ E. coli BW25113 Sourceof ubiC and entC CGSC K. pneumoniae Source of kpdBCD and aroY ATCCPZH572 P. stutzeri OX1 Source of phKLMNOP ATCC P. putida KT2440 Sourceof catA ATCC Plasmid Description Source pTrc99A pBR322 ori Amp^(r),lacIq, P_(trc) Prather Lab, MIT pTrcCOLAK ColA ori, Kan^(r), lacIq,P_(trc) McKenna et al. (2013) pY3 p15A, Amp^(r),lacI,P_(lac)-_(Uv5)-tyrB-tyrA^(fbr)-aroC Juminaga etT1-P_(trc)-aroA-aroL al. (2012) pKD46 repA101(ts) and R101 ori, Amp^(r),araC, araBp CGSC pCP20 FLP, ts-rep, [cI857](lambda)(ts), Amp^(r) CGSCpTutA-Ph tutA of C. braakii inserted to pPh This study pTutA-Ph-CatAtutA of C. braakii inserted to pPh-CatA This study pSDC-PchB-EntC SDC ofT. moniliforme, pchB of P. aeruginosa PAO1, This study and entC of E.coli BW25113 inserted to pTrc99A pUbiC-Kpd ubiC of E. coli BW25113 andkpdBCD of K. pneumoniae This study PZH572 inserted to pTrc99A pPhphKLMNOP of P. stutzeri OX1 inserted to pTrcCOLAK This study pPh-CatAcatA of P. putida KT2440 inserted to pPh This study pUbiC-PobA ubiC ofE. coli BW25113 and pobA of P. aeruginosa Pugh et al. PAO1 inserted topTrc99A (2014) pAroY aroY of K. pneumoniae PHZ572 inserted to pTrcCOLAKPugh et al. (2014) pAroY-CatA catA of P. putida KT2440 inserted to pAroYThis study pQsuB-AroY-CatA qsuB of C. glutamicum inserted to pTrc99AThis study Pathway Plasmid Combination Source CAT2 pY3, pTutA-Ph Thisstudy CAT3 pSDC-PchB-EntC, pPh This study CAT4 pUbiC-Kpd, pPh This studyCAT5 pUbiC-PobA, pAroY Pugh et al. (2014) MA1 pQsuB-AroY-CatA This studyMA2 pY3, pTutA-Ph-CatA This study MA3 pSDC-PchB-EntC, pPh-CatA Thisstudy MA4 pUbiC-Kpd, pPh-CatA This study MA5 pUbiC-PobA, pAroY-CatA Thisstudy MAF pUbiC-PobA, pQsuB-AroY-CatA This study (Pugh, et al., 2014.Process Biochemistry. 49, 1843-1850)

TABLE 2  Primers designed and used in Example 1. Underlinedbases indicate restriction site used for cloning. PrimerSequence (5′ → 3′) SEQ ID NO 32 ATA GGA TCC AGG AGG ATA AAT AAT GCGTAC ATC CAT TGC CAC TGT TTG SEQ ID NO 33ATT AAG CTT CTA GTT TGG GAT TCC CCG CTC GA SEQ ID NO 20ATA CCT GCA GGA GGA GGA TAA ATA ATG ACC GTG AAA ATT TCC CAC ACT GCSEQ ID NO 21 ATA AAG CTT GGA GGA GGA TAA ATA ATGACC GTG AAA ATT TCC CAC ACT GC SEQ ID NO 22ATA GAA TTC AGG AGG ATA AAT AAT GAA TTA TCC GGC AGA ACC SEQ ID NO 23ATT TCT AGA TTA GAT ATA GTC AAA GCG TGC AGT A SEQ ID NO 16ATA GAA TTC AGG AGG ATA AAT AAT GCG TGG TAA AGT TAG CCT G SEQ ID NO 17ATT GGA TCC TTA GGC TTC GCT GTC ATA GAA T SEQ ID NO 14AAT ATC TAG ATT CCC GAG AGG TTG CAT GAT GAA AAC T SEQ ID NO 15ATT GGA TCC TTA TGC GGC ACC CCG TGT CTG G SEQ ID NO 12ATA GGA TCC AGG AGG ATA AAT AAT GGA TAC GTC ACT GGC TGA SEQ ID NO 13ATT CTG CAG TTA ATG CAA TCC AAA AAC GTT SEQ ID NO 24ATA GAA TTC AGG AGG ATA AAT AAT GTC ACA CCC CGC GTT AAC G SEQ ID NO 25ATT AGA TCT TTA GTA CAA CGG TGA CGC CGG TAA A SEQ ID NO 26ATA GGA TCC CCC GTC CGG AGA GGG TAA TTT AAA TAT AAA GTT CG SEQ ID NO 27ATT AAG CTT CTT AGC GGG CCC CTT TAT TAA CGC T SEQ ID NO 18ATA TCT AGA AGG AGG ATA AAT AGA GCT CGT GCT GCC TCA CGA SEQ ID NO 19ATT CCT GCA GGA TGC CCA TGA CTA TAT CTT CTT GAA CAG GGC SEQ ID NO 48CGT GTG AAA CAG AAT GCG AAG ACG AAC AAT A SEQ ID NO 49TAA TCC AGT GCC GGA TGA TTC ACA TCA TC SEQ ID NO 50ATC GCG GCG TTA TTT CAT TCG GAT T SEQ ID NO 51AAC TGT AGG CCG GAT GTG GC SEQ ID NO 52 GCG AGG CAC CAC CAC TTT CGSEQ ID NO 53 AGC GCC CAT CAG GGC G SEQ ID NO 54CTA TGA GCG CCA TTT CTA TCC CGC GC SEQ ID NO 55CCT GAA AGG GAC TGG CGA CCT G

TABLE 3 Catechol production via each of the proposed pathways using E.coli NST74 ΔpheA as host background. Strains were cultured for 120 h andinitially supplied with 20 g/L glucose. Error represents one standarddeviation from triplicate experiments. ^(a)Pugh, et al., 2014. ProcessBiochemistry. 49, 1843-1850. Accumulated Glucose Intermediate(s)Catechol Utilization Y_(P/S) Pathway (mg/L) (mg/L) (%) (mg/g) CAT2 Tyr: 79 ± 3 65 ± 1  6.2 ± 0.28  342 ± 12 Phenol: 149 ± 1 CAT3 Salicylate:100 ± 2 39 ± 1 12.8 ± 0.26  55 ± 2 Phenol: 111 ± 2 CAT4 Phenol:  51 ± 1027 ± 1 9.4 ± 2.1 125 ± 9 CAT5^(a) n.d.  451 ± 44 64 ± 1 35.0 ± 3.0 

TABLE 4 MA production via each of the proposed pathways using E. coliNST74 ΔpheA as host background. Strains were cultured for 120 h andinitially supplied with 20 g/L glucose. Error represents one standarddeviation from triplicate experiments. Accumulated Glucose IntermediateMA utilization Y_(P/S) Pathway Metabolite (mg/L) (mg/L) Acetate (g/L)(%) (mg/g) MA1 n.d. 1586 ± 11  12 ± 0.20 100 ± 1   79.3 ± 0.53 MA2 Tyr: 186 ± 11 10 ± 1.4 44 ± 4 21.0 ± 2.2 220 ± 12 Phenol: 63 ± 1 MA3 n.d. 484 ± 44 7.1 ± 0.12 52 ± 2 46.7 ± 6.0 MA4 n.d.  230 ± 20 7.1 ± 0.13 36± 1 31.8 ± 3.6 MA5 n.d.  819 ± 44  12 ± 0.21 100 ± 1  40.9 ± 2.2 MAFn.d. 2042 ± 88  11 ± 0.17 100 ± 1  102 ± 4.4

TABLE 5 Investigating ‘metabolic funneling’ for improving MA productionusing E. coli NST74 ΔpheA ΔpykA ΔpykF Δcrr as the host background.Strains were cultured for 120 h and initially supplied with 20 g/Lglucose. Error represents one standard deviation from triplicateexperiments. Glucose MA Acetate utilization Y_(P/S) Pathway (mg/L) (g/L)(%) (mg/g) MA1 1792 ± 28 n.d. 100 ± 1 89.6 ± 1.4 MAF  3153 ± 149 7 ± 0.1100 ± 1  158 ± 7.4

TABLE 6 Strains, plasmids, and pathways constructed and/or used inExample 2. Strain Description Source E. coli NEB10-beta Δ(ara-leu) 7697araD139 fhuA ΔlacX74 galK16 NEB galE15 e14- φ80dlacZΔM15 recAl relAlendAl nupG rpsL (Str^(R)) rph spoT1 Δ(mrr-hsdRMS-mcrBC) E. coli BW25113Source of ubiC and entC CGSC E. coli JW2580-1 Source of pheA::Kan^(R)CGSC E. coli JW1843-2 Source of pykA::Kan^(R) CGSC E. coli JW1666-3Source of pykF::Kan^(R) CGSC E. coli JW2410-1 Source of crr::Kan^(R)CGSC E. coli NST74 aroH367, tyrR366, tna-2, lacY5, aroF394(fbr), ATCCmalT384, pheA101(fbr), pheO352, aroG397(fbr) E. coli NST74 ΔpheA pheAchromosomal deletion in E. coli NST74 Pugh et al. (2014) E. coli NST74ΔpheA crr chromosomal deletion in E. coli NST74 ΔpheA This study ΔpykAΔpykF Δcrr ΔpykA ΔpykF C. braakii Source of tutA ATCC P. aeruginosa PAO1Source of pchB DSMZ E. coli BW25113 Source of ubiC and entC CGSC K.pneumoniae Source of kpdBCD ATCC PZH572 Plasmid Description SourcepTrcCOLAK ColA ori, Kan^(r), lacIq, P_(trc) McKenna et al. (2013) pY3p15A, Amp^(r), lacI, P_(lac-UV5)-tyrB-tyrA^(fb)r-aroC Juminaga etT1-P_(trc)-aroA-aroL al. (2012) pS3 pBBR1 ori; Cm^(r), lacI Juminaga etP_(lac-UV5)-aroE-aroD-aroB^(op)-aroG^(fbr)-ppsA-tktA al. (2012) pKD46repA101(ts) and R101 ori, Amp^(r), araC, araBp CGSC pCP20 FLP, ts-rep,[cI857](lambda)(ts), Amp^(r) CGSC pTyrAfbr-TutA tyrA^(fbr) of E. coliand tutA of C. braakii inserted This study to pY3 backbonepSDC-PchB-EntC SDC of T. moniliforme, pchB of P. aeruginosa PAO1, Thisstudy and entC of E. coli BW25113 inserted to pS3 backbone pUbiC-KpdubiC of E. coli and kpdBCD of K. pneumoniae Thompson et PZH572 insertedto pTrcCOLAK al. (2016) (Pugh, et al., 2014. Process Biochemistry. 49,1843-1850; Thompson, et al., 2016. Biotechnol Bioeng. 113 (8), 1745-54)

TABLE 7 Primers designed and used in Example 2. Underlinedbases indicate restriction site used for cloning. PrimerSequence (5′ → 3′) SEQ ID NO 22 ATA GAA TTC AGG AGG ATA AAT AATGAA TTA TCC GGC AGA ACC SEQ ID NO 23 ATT TCT AGA TTA GAT ATA GTC AAAGCG TGC AGT A SEQ ID NO 14 AAT ATC TAG ATT CCC GAG AGG TTGCAT GAT GAA AAC T SEQ ID NO 15 ATT GGA TCC TTA TGC GGC ACC CCG TGT CTG GSEQ ID NO 12 ATA GGA TCC AGG AGG ATA AAT AAT GGA TAC GTC ACT GGC TGASEQ ID NO 13 ATT CTG CAG TTA ATG CAA TCC AAA AAC GTT SEQ ID NO 16ATA CCA TGG AGG AGG ATA AAT AAT GCG TGG TAA AGT TAG CCT G SEQ ID NO 17ATT GGA TCC TTA GGC TTC GCT GTC ATA GAA T SEQ ID NO 24ATA GAA TTC AGG AGG ATA AAT AAT GTC ACA CCC CGC GTT AAC G SEQ ID NO 25ATT AGA TCT TTA GTA CAA CGG TGA CGC CGG TAA A SEQ ID NO 26ATA GGA TCC CCC GTC CGG AGA GGG TAA TTT AAA TAT AAA GTT CG SEQ ID NO 27ATT AAG CTT CTT AGC GGG CCC CTT TAT TAA CGC T SEQ ID NO 34AGA TCT AAA GGA GGC CAT CCA TGG CTG GAA ACA CAA TTG G SEQ ID NO 35ATG CCT GGA GAT CCT TAC TCG AGT TTG GAT CCT C SEQ ID NO 36GAG GAT CCA AAC TCG AGT AAG GAT CTC CAG GCA T SEQ ID NO 37CCA ATT GTG TTT CCA GCC ATG GAT GGC CTC CTT TAG ATC T SEQ ID NO 38CTG CAC GCT TTG ACT ATA TCT AAG GAT CCA AAC TCG AGT AAG G SEQ ID NO 39CAT GGA TGG CCT CCT AGA TCT TTT GAA TTC TGA AAT TGT TAT C SEQ ID NO 40GAT AAC AAT TTC AGA ATT CAA AAG ATC TAG GAG GCC ATC CAT G SEQ ID NO 41CGG ATA ATT CAT TAT TTA TCC TCC TTT AGA TCC TTA CTG GCG ATT SEQ ID NO 42AAT CGC CAG TAA GGA TCT AAA GGA GGA TAA ATA ATG AAT TAT CCG SEQ ID NO 43CCT TAC TCG AGT TTG GAT CCT TAG ATA TAG TCA AAG CGT GCA G SEQ ID NO 44AAG GAG GCC ATC CAT GCG TGG TAA AGT TAG C SEQ ID NO 45GTT TGG ATC CTT AAT GCA ATC CAA AAA CG SEQ ID NO 46ATT GCA TTA AGG ATC CAA ACT CGA GTA AG SEQ ID NO 47CTT TAC CAC GCA TGG ATG GCC TCC TTT AGA TC

TABLE 8 Pathways Constructed for Phenol Production via Synthetic‘Metabolic Funneling’. Pathway Plasmid Combination Source  PH1pTyrAfbr-TutA, This study  PH2 pSDC-PchB-EntC This study  PH3 pUbiC-KpdThis study PHF1 pTyrAfbr-TutA, pSDC-PchB-EntC This study PHF2pTyrAfbr-TutA, pUbiC-Kpd This study PHF3 pSDC-PchB-EntC, pUbiC-Kpd Thisstudy PHF4 pTyrAfbr-TutA, pSDC-PchB- This study EntC, pUbiC-Kpd

TABLE 9 Evaluating the Effects of Synthetic ‘Metabolic Funneling’ forPhenol Biosynthesis. All pathways were expressed using E. coli NST74ΔpheA as host background. Strains were cultured fbr 120 h and initiallysupplied with 20 g/L glucose. Error represents one standard deviationfrom triplicate experiments. Glucose Phenol Utilization Acetate Y_(P/S)Pathway (mg/L) (%) (g/L) (mg/g)  PH1 377 ± 23 100 ± 1 9 ± 0.1 18.7 ±0.72  PH2 377 ± 14   53 ± 0.7 6 ± 0.3 35.7 ± 0.80  PH3 149 ± 12 100 ± 14 ± 0.3  7.3 ± 0.31 PHF1 439 ± 7    90 ± 0.9 8 ± 0.2 24.0 ± 0.51 PHF2355 ± 17 100 ± 1 6 ± 0.2 17.8 ± 1.2  PHF3 149 ± 10   90 ± 0.4 6 ± 0.38.4 ± 1.0 PHF4 205 ± 13   87 ± 0.3 5 ± 0.2 11.8 ± 0.72

TABLE 10 Comparing Engineering Strategies for Phenol Production. Allpathways were expressed using E. coli NST74 ΔpheA ΔpykA ΔpykF Δcrr asthe host background. Strains were cultured for 120 h and initiallysupplied with 20 g/L glucose. Error represents one standard deviationfrom triplicate experiments. Glucose Phenol Utilization Acetate Y_(P/S)Pathway (mg/L) (%) (g/L) (mg/g)  PH1 329 ± 9  100 ± 1 6 ± 0.1 16.4 ±0.65  PH2 277 ± 15 100 ± 1 7 ± 0.1 13.8 ± 0.19 PHF1 575 ± 19 100 ± 1 5 ±0.3 28.8 ± 0.34

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andillustrative embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

What is claimed is:
 1. A method for preparing a biochemical product, the method comprising: i) contacting a recombinant host cell with a fermentable carbon source, wherein the recombinant host comprises two or more recombinant pathways, and wherein: a) each pathway is capable of producing the same final biochemical product; b) each pathway comprises at least one gene encoding a polypeptide; c) each pathway is derived from a different endogenous metabolite as its immediate precursor; and d) each pathway converges to the same final product or the same intermediate metabolite; and ii) growing said recombinant cell for a time sufficient to synthesize the product.
 2. The method of claim 1, wherein each pathway converges to the same final product.
 3. The method of claim 1, wherein each pathway converges to the same intermediate metabolite, further comprising each pathway continuing to the same final product.
 4. A method for preparing a biochemical product, the method comprising: i) contacting a recombinant host cell with a fermentable carbon source, wherein the recombinant host comprises two or more recombinant pathways, and wherein: a) each pathway is capable of producing the same final biochemical product; b) each pathway comprises at least one gene encoding a polypeptide; c) each pathway is derived from the same endogenous metabolite as its immediate precursor; d) each pathway proceeds via different intermediate metabolites; e) each pathway comprises at least one gene encoding polypeptides with differing activities; and f) each pathway converges to the same final product or the same intermediate metabolite; and ii) growing said recombinant cell for a time sufficient to synthesize the product.
 5. The method of claim 4, wherein each pathway converges to the same final product.
 6. The method of claim 4, wherein each pathway converges to the same intermediate metabolite, further comprising each pathway continuing to the same final product.
 7. The method of any one of claims 1-6, wherein the biochemical product is phenol.
 8. The method of claim 7, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having isochorismate synthase activity; b) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; c) at least one gene encoding a polypeptide having salicylate decarboxylase activity; and d) at least one gene encoding a polypeptide having tyrosine phenol lyase activity.
 9. The method of claim 7, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; and c) at least one gene encoding a polypeptide having tyrosine phenol lyase activity.
 10. The method of claim 7, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having isochorismate synthase activity; b) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; c) at least one gene encoding a polypeptide having salicylate decarboxylase activity; d) at least one gene encoding a polypeptide having chorismate lyase activity; and e) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity.
 11. The method of claim 7, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having isochorismate synthase activity; b) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; c) at least one gene encoding a polypeptide having salicylate decarboxylase activity; d) at least one gene encoding a polypeptide having chorismate lyase activity; e) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; and f) at least one gene encoding a polypeptide having tyrosine phenol lyase activity.
 12. The method of anyone of claims 1-6, wherein the biochemical product is catechol.
 13. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having isochorismate synthase activity; b) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; c) at least one gene encoding a polypeptide having salicylate decarboxylase activity; d) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; and e) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 14. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; c) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; and d) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 15. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having isochorismate synthase activity; b) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; c) at least one gene encoding a polypeptide having salicylate decarboxylase activity; d) at least one gene encoding a polypeptide having chorismate lyase activity; e) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; and f) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 16. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; c) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; d) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; and e) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 17. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; c) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; d) at least one gene encoding a polypeptide having isochorismate synthase activity; e) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; f) at least one gene encoding a polypeptide having salicylate decarboxylase activity; and g) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 18. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; c) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; d) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; and e) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 19. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having chorismate lyase activity; c) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; and d) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity.
 20. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; c) at least one gene encoding a polypeptide having chorismate lyase activity; d) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; and e) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 21. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; c) at least one gene encoding a polypeptide having isochorismate synthase activity; d) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; e) at least one gene encoding a polypeptide having salicylate decarboxylase activity; and f) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 22. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; c) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; and d) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 23. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having chorismate lyase activity; c) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; d) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; e) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; and f) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 24. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having chorismate lyase activity; c) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; d) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; e) at least one gene encoding a polypeptide having isochorismate synthase activity; f) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; g) at least one gene encoding a polypeptide having salicylate decarboxylase activity; and h) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 25. The method of claim 12, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having chorismate lyase activity; c) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; d) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; e) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; and f) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 26. The method of any one of claims 1-6 wherein the biochemical product is muconic acid or a salt thereof.
 27. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having isochorismate synthase activity; b) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; c) at least one gene encoding a polypeptide having salicylate decarboxylase activity; d) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; e) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and f) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 28. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; c) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; d) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and e) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 29. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having isochorismate synthase activity; b) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; c) at least one gene encoding a polypeptide having salicylate decarboxylase activity; d) at least one gene encoding a polypeptide having chorismate lyase activity; e) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; f) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and g) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 30. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; c) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; d) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; e) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and f) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 31. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; c) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; d) at least one gene encoding a polypeptide having isochorismate synthase activity; e) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; f) at least one gene encoding a polypeptide having salicylate decarboxylase activity; g) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and h) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 32. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; c) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; d) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; e) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and f) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 33. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having chorismate lyase activity; c) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; d) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; and e) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 34. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; c) at least one gene encoding a polypeptide having chorismate lyase activity; d) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; e) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and f) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 35. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; c) at least one gene encoding a polypeptide having isochorismate synthase activity; d) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; e) at least one gene encoding a polypeptide having salicylate decarboxylase activity; f) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and g) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 36. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; c) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; d) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and e) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 37. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having chorismate lyase activity; c) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; d) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; e) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; f) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and g) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 38. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having chorismate lyase activity; c) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; d) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; e) at least one gene encoding a polypeptide having isochorismate synthase activity; f) at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity; g) at least one gene encoding a polypeptide having salicylate decarboxylase activity; h) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and i) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 39. The method of claim 26, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity; b) at least one gene encoding a polypeptide having chorismate lyase activity; c) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; d) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; e) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; f) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and g) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 40. A method for preparing phenol, the method comprising: i) contacting a recombinant host cell with a fermentable carbon source, said recombinant host comprising: a) at least one gene encoding a polypeptide having chorismate lyase activity; and b) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; and ii) growing said recombinant cell for a time sufficient to produce phenol.
 41. A method for preparing catechol, the method comprising: i) contacting a recombinant host cell with a fermentable carbon source; and ii) growing said recombinant cell for a time sufficient to produce catechol.
 42. The method of claim 41, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; and c) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 43. The method of claim 41, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; and b) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity.
 44. The method of claim 41, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; and c) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity.
 45. A method for preparing muconic acid, the method comprising: i) contacting a recombinant host cell with a fermentable carbon source; and ii) growing said recombinant cell for a time sufficient to produce muconic acid.
 46. The method of claim 45, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity; c) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and d) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 47. The method of claim 45, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having tyrosine phenol lyase activity; b) at least one gene encoding a polypeptide having phenol 2-monooxygenase activity; and c) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 48. The method of claim 45, wherein the recombinant host comprises: a) at least one gene encoding a polypeptide having chorismate lyase activity; b) at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity; c) at least one gene encoding a polypeptide having protocatechuate decarboxylase activity; and d) at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity.
 49. The method of any one of claims 1-48, wherein the fermentable carbon source is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, glycerol, carbon dioxide, methanol, methane, formaldehyde, formate, amino acids, carbon-containing amines.
 50. The method of any one of claims 1-48, wherein the fermentable carbon source is glucose, xylose or glycerol.
 51. The method of any one of claims 1-50, wherein: a) the at least one gene encoding a polypeptide having isochorismate synthase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:1; b) the at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:2; c) the at least one gene encoding a polypeptide having salicylate decarboxylase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:3; d) the at least one gene encoding a polypeptide having phenol 2-monooxygenase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:4; e) the at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:5. f) the at least one gene encoding a polypeptide having tyrosine phenol lyase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:6; g) the at least one gene encoding a polypeptide having chorismate lyase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:7; h) the at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:8; i) the at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:9; j) the at least one gene encoding a polypeptide having protocatechuate decarboxylase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:10; and/or k) the at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity comprises a sequence having at least about 70% sequence identity to SEQ ID NO:11.
 52. The method of any one of claims 1-50, wherein: a) the at least one gene encoding a polypeptide having isochorismate synthase activity is entC, menF, pchA or ICS1; b) the at least one gene encoding a polypeptide having isochorismate pyruvate lyase activity is pchB; c) the at least one gene encoding a polypeptide having salicylate decarboxylase activity is SDC; d) the at least one gene encoding a polypeptide having phenol 2-monooxygenase activity is dmpLMNOP or phKLMNOP; e) the at least one gene encoding a polypeptide having 1,2-catechol dioxygenase activity is catA or salD. f) the at least one gene encoding a polypeptide having tyrosine phenol lyase activity is tutA; g) the at least one gene encoding a polypeptide having chorismate lyase activity is ubiC; h) the at least one gene encoding a polypeptide having p-hydroxybenzoate decarboxylase activity is kpdBCD; i) the at least one gene encoding a polypeptide having p-hydroxybenzoate hydroxylase activity is pobA; j) the at least one gene encoding a polypeptide having protocatechuate decarboxylase activity is aroY; and/or k) the at least one gene encoding a polypeptide having 3-dehydroshikimate dehydratase activity is aroZ, quiC or qsuB.
 53. A recombinant host cell as described in any one of claims 1-52.
 54. The method or recombinant host of any one of claims 1-53, wherein the recombinant host cell is selected from the group consisting of bacteria, yeast, filamentous fungi, cyanobacteria, algae, and plant cells.
 55. The method or recombinant host cell of any one of claims 1-53, wherein the recombinant host cell is selected from the group consisting of Escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Sphingomonas, Yarrowia, Clostridium, Corynebacterium, Methylosinus, Methylomonas, Rhodococcus, Pseudomonas, Rhodobacter, Synechocystis, Saccharomyces, Klebsiella, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Torulopsis, Aspergillus, Arthrobotrys, Brevibacterium, Microbacterium, Arthrobacter, Ctirobacter, Chlamydomonas, and Zymomonas.
 56. The method or recombinant host cell of claim 55, wherein the recombinant host cell is E. coli NST74, E. coli NST74 ΔpheA, E. coli NST74 ΔpheA ΔpykA ΔpykF or E. coli NST74 ΔpheA ΔpykA ΔpykF Δcrr.
 57. The method or recombinant host cell of any one of claims 1-56, wherein the recombinant host cell comprises a plasmid combination selected from the group consisting of: pY3 and pTutA-pPh; pSDC-PchB-EntC and pPh; pUbiC-Kpd and pPh; pUbiC-PobA and pAroY; pQsuB-AroY-CatA; pY3 and pTutA-pPh-CatA; pSDC-PchB-EntC and pPh-CatA; pUbiC-Kpd and pPh-CatA; pUbiC-PobA and pAroY-CatA; and pUbiC-PobA; pQsuB-AroY-CatA; pTyrAfbr-TutA; pSDC-PchB-EntC; pUbiC-Kpd; pTyrAfbr-TutA and pSDC-PchB-EntC; pTyrAfbr-TutA and pUbiC-Kpd; pSDC-PchB-EntC and pUbiC-Kpd; and pTyrAfbr-TutA, pSDC-PchB-EntC and pUbiC-Kpd. 